Combined tissue factor and chemotherapeutic methods and compositions for coagulation and tumor treatment

ABSTRACT

The invention embodies the surprising discovery that Tissue Factor (TF) compositions and variants thereof specifically localize to the blood vessels within a vascularized tumor following systemic administration. The invention therefore provides methods and compositions comprising coagulation-deficient Tissue Factor for use in effecting specific coagulation and for use in tumor treatment. The TF compositions and methods of present invention may be used alone, as TF conjugates with improved half-life, or in combination with other agents, such as conventional chemotherapeutic drugs, targeted immunotoxins, targeted coaguligands, and/or in combination with Factor VIIa (FVIIa) or FVII activators.

The U.S. Government owns rights in the present invention pursuant togrant numbers ROI-CA59569, ROI-CA54168 and POI-HL16411 from the NationalInstitutes of Health.

The present application is a non-provisional application and claims thebenefit of provisional application Serial No. 60/042,427, filed Mar. 27,1997; provisional application Serial No. 60/036,205, filed Jan. 27,1997; and provisional application Serial No. 60/035,920, filed Jan. 22,1997; the entire disclosures of each of which provisional applicationsare incorporated herein by reference without disclaimer.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the fields of blood vesselsand of coagulation. More particularly, it embodies the surprisingfinding that Tissue Factor compositions can localize to tumorvasculature and cause specific coagulation. Methods and compositions foreffecting specific coagulation and for treating tumors with modifiedTissue Factor (TF) compositions and combinations of TF and othermolecules are particularly provided.

2. Description of Related Art

Tumor cell resistance to various chemotherapeutic agents represents amajor problem in clinical oncology. Therefore, although many advances inthe chemotherapy of neoplastic disease have been realized during thelast 30 years, many of the most prevalent forms of human cancer stillresist effective chemotherapeutic intervention.

A significant underlying problem that must be addressed in any treatmentregimen is the concept of "total cell kill." This concept holds that inorder to have an effective treatment regimen, whether it be a surgicalor chemotherapeutic approach or both, there must be a total cell kill ofall so-called "clonogenic" malignant cells, that is, cells that have theability to grow uncontrolled and replace any tumor mass that might beremoved. Due to the ultimate need to develop therapeutic agents andregimens that will achieve a total cell kill, certain types of tumorshave been more amenable than others to therapy. For example, the softtissue tumors (e.g., lymphomas), and tumors of the blood andblood-forming organs (e.g., leukemias) have generally been moreresponsive to chemotherapeutic therapy than have solid tumors such ascarcinomas.

One reason for the susceptibility of soft and blood-based tumors tochemotherapy is the greater physical accessibility of lymphoma andleukemic cells to chemotherapeutic intervention. Simply put, it is muchmore difficult for most chemotherapeutic agents to reach all of thecells of a solid tumor mass than it is the soft tumors and blood-basedtumors, and therefore much more difficult to achieve a total cell kill.Increasing the dose of chemotherapeutic agents most often results intoxic side effects, which generally limits the effectiveness ofconventional anti-tumor agents.

It has long been quite clear that a significant need exists for thedevelopment of novel strategies for the treatment of solid tumors. Onesuch strategy is the use of "immunotoxins", in which an anti-tumor cellantibody is used to deliver a toxin to the tumor cells. However, incommon with the chemotherapeutic approach described above, this alsosuffers from certain drawbacks. For example, antigen-negative orantigen-deficient cells can survive and repopulate the tumor or lead tofurther metastases. Also, in the treatment of solid tumors, the tumormass is generally impermeable to molecules of the size of the antibodiesand immunotoxins. Therefore, the development of immunotoxins alone didnot lead to particularly significant improvements in cancer treatment.

Certain investigators then developed the approach of targeting thevasculature of solid tumors. Targeting the blood vessels of the tumorshas certain advantages in that it is not likely to lead to thedevelopment of resistant tumor cells or populations thereof.Furthermore, delivery of targeted agents to the vasculature does nothave problems connected with accessibility, and destruction of the bloodvessels should lead to an amplification of the anti-tumor effect as manytumor cells rely on a single vessel for their oxygen and nutrientsupplies. Exemplary vascular targeting strategies are described inBurrows et al. (1992), in Burrows and Thorpe (1993) and in WO 93/17715.Such targeted delivery of anti-cellular agents to tumor vasculatureprovides quite promising strategies, however, the use of the toxinportions of these molecules still leaves room for improvement invascular targeting.

Another approach for the targeted destruction of tumor vasculature hasbeen reported in WO 96/01653, in which antibodies against tumorvasculature markers are used to deliver coagulants to the vasculature ofsolid tumors. The targeted delivery of coagulants in this manner has theadvantage that significant toxic side effects are not likely to resultfrom any background mis-targeting that may result due to any low levelcross-reactivity of the targeting antibodies with the cells of normaltissues. The antibody-coagulant constructs for use in such directedanti-tumor therapy have been termed "coaguligands" (WO 96/01653).

Although the specific delivery of a coagulant to a tumor vessel marks asurprising advance, the use or manipulation of coagulation in connectionwith treatment of various human diseases and disorders has beenpracticed for some time. By way of example only, Morrissey and Comp haveproposed the use of truncated Tissue Factor (tTF) in combination withFactor VIIa (FVIIa) in the treatment of patients, such as hemophiliacs,in which blood coagulation is impaired (U.S. Pat. Nos. 5,374,617;5,504,064; and 5,504,067). Roy and Vehar have also developed TissueFactor mutants that neutralize endogenous Tissue Factor and may be usedas anti-coagulants, e.g., in the treatment of myocardial infarction(U.S. Pat. Nos. 5,346,991 and 5,589,363).

In further studies connected with Tissue Factor (TF), Edgington andcolleagues have shown that, in contrast to normal melanocytes, malignantmetastasizing human melanoma cells express high levels of TF, the majorcellular initiator of the plasma coagulation protease cascades (WO94/28017; WO 94/05328; U.S. Pat. No. 5,437,864). It was reported thatinhibition of TF function and subsequent reduction in local proteasegeneration resulted in significantly reduced numbers of tumor cellsretained in the vasculature. This led to the suggestion that there was adirect correlation between TF expression and the metastatic phenotype oftumor cells. Edgington and colleagues proposed that a function of TF isrequired for successful implantation of tumor cells and thatinterference with TF function, or specific interference with cellsurface expression of TF, is useful in inhibiting metastasis. Theseauthors have therefore proposed treating cancer with antibodies directedagainst Tissue Factor.

SUMMARY OF THE INVENTION

In direct contrast to the above observations of Edgington and colleaguesand the uses of anti-TF antibodies to treat cancer, the presentinventors have demonstrated that truncated TF compositions and TFvariants can, themselves, be employed in the treatment of solid tumors.The present invention was developed, in part, from the inventors'surprising discovery that truncated TF specifically localizes to theblood vessels within a vascularized tumor simply following systemicadministration. This localization in the absence of any targeting moietycould not have been predicted from the previous detailed studies of theTF molecule. The self-localizing nature of TF, as described herein, alsocontrasts with the previously described uses of TF in the treatment ofbleeding disorders, e.g., in hemophiliacs, in which TF delivery andaction is either not localized or is limited to topical application to aspecific area.

Therefore, in certain embodiments, the present invention providesmethods for promoting coagulation in prothrombotic blood vessels of ananimal or patient, which methods generally comprise administering to theanimal a composition comprising a coagulation-deficient Tissue Factor(TF) compound in an amount effective to promote coagulationpreferentially, or specifically, in the prothrombotic blood vessels.

As used throughout the entire application, the terms "a" and "an" areused in the sense that they mean "at least one", "at least a first","one or more" or "a plurality" of the referenced components or steps,except in instances wherein an upper limit is thereafter specificallystated. Therefore "a coagulation-deficient Tissue Factor" means "atleast a first coagulation-deficient Tissue Factor". The operable limitsand parameters of combinations, as with the amounts of any single agent,will be known to those of ordinary skill in the art in light of thepresent disclosure.

The prothrombotic blood vessels may be associated with any one of avariety of angiogenic diseases, with a benign growth or with avascularized tumor. In the context of the present invention, the term "avascularized tumor" means a vascularized, malignant tumor. The presentinvention is particularly advantageous in treating vascularized tumorsof at least about medium size and in treating large vascularized tumors.

The composition will generally be pharmaceutically acceptable and willpreferably be administered to the animal systemically, such as viaintravenous injection.

The methods of the invention are further described as methods fortreating an animal or human patient having a disease associated withprothrombotic blood vessels, comprising administering to the animal anamount of at least a first coagulating composition comprising at least afirst coagulation-deficient Tissue Factor compound effective topreferentially, or specifically, promote coagulation in theprothrombotic blood vessels associated with the benign or malignantdisease site.

The essence of the invention may also be defined as a compositioncomprising at least a biologically effective amount of at least a firstcoagulation-deficient Tissue Factor compound for use in the preparationof a medicament for use in promoting coagulation preferentially, orspecifically, in prothrombotic blood vessels of an animal, particularlythose associated with a benign or malignant disease site.

In the methods, medicaments and uses of the present invention, one ofthe advantages lies in the fact that the simple provision of thecoagulating composition into the systemic circulation of the animalresults in the specific or preferential localization of the TissueFactor compound to the disease site.

Preferred methods disclosed herein are those for use in promotingcoagulation in the tumor vasculature of an animal or human subjecthaving a vascularized tumor, which methods generally compriseadministering to the animal one or more compositions comprising one ormore coagulation-deficient Tissue Factor compounds in an amountsufficient to specifically or preferentially promote coagulation in thetumor vasculature. The treatment of mid-size or large vascularizedtumors is particularly advantageous.

The treatment methods may be described as methods for treating an animalhaving a vascularized tumor, comprising administering to the animal abiologically effective amount of at least one coagulating compositionthat comprises an amount of at least a first coagulation-deficientTissue Factor compound sufficient to specifically or preferentiallypromote coagulation in the vasculature of the tumor.

A further description is of a method for treating an animal or patienthaving a vascularized tumor which comprises systemically administeringto the animal one or more compositions comprising one or a plurality ofcoagulation-deficient Tissue Factor compounds in an amount(s) and for aperiod of time(s) effective to promote coagulation specifically orpreferentially in the vasculature of the vascularized tumor.

The anti-tumor effects of the present invention are particularlydescribed in the methods characterized as comprising administering to ananimal with a tumor a composition comprising at least onecoagulation-deficient Tissue Factor compound in an amount effective topromote coagulation in the tumor vasculature and to specifically orpreferentially cause tissue necrosis in the tumor.

These aspects of the invention also provide a composition comprising atleast a biologically effective amount of at least a firstcoagulation-deficient Tissue Factor compound for use in the preparationof a medicament for use in promoting coagulation preferentially, orspecifically, in the prothrombotic blood vessels associated with amalignant, vascularized tumor of an animal; wherein the medicament isthus intended for use in treating an animal with cancer by causing tumorblood vessel coagulation and tumor necrosis.

The terms "preferentially" and "specifically", as used herein in thecontext of promoting coagulation in prothrombotic blood vessels or tumorvasculature, and/or as used in the context of promoting coagulationsufficient to cause tissue necrosis in a disease site such as a tumor,mean that the Tissue Factor compound or TF-second agent combinationfunctions to achieve coagulation and/or tissue necrosis that issubstantially confined to the disease site, such as the tumor region,and does not substantially extend to causing coagulation or tissuenecrosis in normal, healthy tissues.

The coagulation-deficient Tissue Factor compound or combinations thereofthus exert coagulative and/or tissue destructive effects in a disease ortumor site and yet have little or no coagulative or tissue destructiveeffects on normal, healthy cells or tissues. Coagulation and/or tissuedestruction is therefore localized to the disease or tumor site and doesnot substantially or significantly extend to other major or importantblood vessels or tissues. In the methods of the invention the functionof healthy cells and tissues is therefore maintained substantiallyunimpaired.

The "coagulation-deficient Tissue Factors" of the invention willgenerally be Tissue Factor compounds that are at least about 100-foldless active than full length, native Tissue Factor, e.g., when assayedin an appropriate phospholipid environment. The Tissue Factor compoundswill still have activity, and are preferably described as being betweenabout 100-fold and about 1,000,000 less active than full length, nativeTissue Factor, e.g., when assayed in an appropriate phospholipidenvironment.

The coagulation-impaired Tissue Factor compounds will preferably be atleast about 1,000-fold less active than full length, native TissueFactor; more preferably will be at least about 10,000-fold less activethan full length, native Tissue Factor; even most preferably will be atleast about 100,000-fold less active than full length, native TissueFactor, e.g., when assayed in an appropriate phospholipid environment.

The "at least about 100,000-fold less active" is not the minimum, andthe Tissue Factor compounds may be at least about 500,000-fold or about1,000,000-fold less active than full length, native Tissue Factor, e.g.,when assayed in an appropriate phospholipid environment.

The human Tissue Factor compounds will generally be preferred for humanuses, but the use of other species of TF, including E. coli TF, iscertainly not excluded. For ease of preparation, thecoagulation-deficient Tissue Factor compounds will also preferably beprepared by recombinant expression, although this is not essential.

The Tissue Factor may be rendered coagulation deficient by beingdeficient in binding to a phospholipid surface and/or deficient ininserting into a phospholipid membrane or lipid bilayer. Preferredexamples are "truncated Tissue Factors". As defined in U.S. Pat. No.5,504,064, in which the compounds are used for different purposes,"truncated Tissue Factors" generally have an amino acid sequencediffering from that of native Tissue Factor in that sufficienttransmembrane amino acids that function to bind native Tissue Factor tophospholipid membranes are lacking from the truncated Tissue Factorprotein so that the truncated Tissue Factor protein does not bind tophospholipid membranes.

Particular examples of truncated Tissue Factors are Tissue Factorcompounds comprising about the first 219 contiguous amino acids from thenative TF sequence, as further exemplified by a Tissue Factor compoundthat consists essentially of the amino acid sequence of SEQ ID NO:1.Although intended for use in different methods, U.S. Pat. No. 5,504,067defines truncated Tissue Factors as Tissue Factor proteins having anamino acid sequence beginning at position 1 and terminating nearposition 219 of the defined Tissue Factor sequence.

Dimeric coagulation-deficient Tissue Factors may also be employed,including homodimeric and heterodimeric Tissue Factors. Exemplary TFdimers are disclosed herein as those that consist essentially of dimersof the amino acid sequence of SEQ ID NO:3 (H₆ -tTF₂₁₉ -cys-C' dimer),SEQ ID NO:6 (H₆ -tTF₂₂₀ -cys-C' dimer), SEQ ID NO:7 (H₆ -tTF₂₂₁ -cys-C'dimer) or SEQ ID NO:2 (H₆ -N'-cys-tTF₂₁₉ dimer). Chemically conjugateddimers, as described in detail hereinbelow, are preferred for use incertain aspects of the present invention, although recombinantlyproduced dimers, in frame with in frame linkers, are also contemplatedfor use in particular embodiments.

The coagulation-impaired Tissue Factor compounds for use herewith mayalso be polymeric or multimeric Tissue Factors.

In certain embodiments, the Tissue Factor compound will be a mutantTissue Factor deficient in the ability to activate Factor VII. Althoughuseful alone, the most preferred uses of such mutants will be inconjunction with the co-administration of a biologically effectiveamount of at least one of Factor VIIa or an activator of Factor VII,such as when used with an amount of Factor VIIa sufficient to increasetumor vasculature coagulation and tumor necrosis in the animal.

Such mutants may be those that include a mutation in the amino acidregion between about position 157 and about position 167 of SEQ ID NO:1.Exemplary, but by no means limiting mutants are those wherein, withinSEQ ID NO:1, Trp at position 158 is changed to Arg; wherein Ser atposition 162 is changed to Ala; wherein Gly at position 164 is changedto Ala; or wherein Trp at position 158 is changed to Arg and Ser atposition 162 is changed to Ala. Defined examples of such mutants arethose that consist essentially of the amino acid sequence of SEQ ID NO:8or SEQ ID NO:9.

Any of the truncated, dimeric, multimeric and/or mutantcoagulation-deficient Tissue Factor compounds may further be modified toincrease the longevity, half life or "biological half life" of the TFmolecule. Various modifications of the polypeptide structure may be madein order to effect such a change in properties.

Particular examples of TFs modified to increase their biological halflife are those Tissue Factor compounds that have been operativelyattached, and preferably covalently linked, to a carrier molecule, suchas a protein carrier. The carriers are preferably inert carriers, suchas, by way of example only, an albumin or a globulin. Non-proteincarriers such as polysaccharides and synthetic polymers are alsocontemplated.

The operative attachment of a TF construct to an antibody or portionthereof is a currently preferred form of coagulation-deficient TF withincreased biological half life. However, in the context of the firstagent for use in the anti-cancer treatment strategies provide herein,the TF will be linked to an antibody that does not exhibit significantspecific binding to a component of a tumor cell, tumor vasculature ortumor stroma. That is, wherein the Tissue Factor compound is notattached to an "anti-tumor" antibody, and wherein the resultant TissueFactor compound is not a "tumor-targeted TF compound".

In such TF-antibody conjugates, the Tissue Factor compound may beoperatively attached to an IgG molecule of so-called "irrelevantspecificity", i.e., one that does not have immunobinding affinity for acomponent of a tumor cell, tumor vasculature or tumor stroma. The TissueFactor compounds may equally be operatively attached to an Fc portion ofan antibody, which has no specific targeting function in the context ofantibody specificity. Further constructs contemplated are those whereinthe Tissue Factor compound has been introduced into an IgG molecule inplace of the C_(H) 3 domain.

The surprisingly effective TF treatments of the present invention may beadvantageously combined with one or more other treatments. For example,the treatment methods may further comprise administering to an animal orpatient a biologically or therapeutically effective amount of at least asecond therapeutic compound, such as at least one of a secondtherapeutic compound selected from the group consisting of Factor VIIa,an activator of Factor VII and at least a first anti-cancer agent.

The at least a first anti-cancer agent may be a "chemotherapeuticagent". As used herein, the term "chemotherapeutic agent" is used torefer to a classical chemotherapeutic agent or drug used in thetreatment of malignancies. This term is used for simplicitynotwithstanding the fact that other compounds, including immunotoxins,may be technically described as a chemotherapeutic agent in that theyexert an anti-cancer effect. However, "chemotherapeutic" has come tohave a distinct meaning in the art and is being used according to thisstandard meaning. "Chemotherapeutics" in the context of the presentapplication therefore do not generally refer to immunotoxins,radiotherapeutic agents and such like, despite their operationaloverlap.

A number of exemplary chemotherapeutic agents are listed in Table II.Those of ordinary skill in the art will readily understand the uses andappropriate doses of chemotherapeutic agents, although the doses maywell be reduced when used in combination with the present invention. Acurrently preferred chemotherapeutic agent is etoposide. A new class ofdrugs that may also be termed "chemotherapeutic agents" are agents thatinduce apoptosis. Any one or more of such drugs, including genes,vectors and antisense constructs, as appropriate, may also be used inconjunction with the present invention.

Appropriate anti-cancer agents further include specifically targetedtoxic agents. For example, anti-cancer antibodies and, preferably,antibody constructs or conjugates comprising an antibody thatspecifically binds to a component of a tumor cell, tumor vasculature ortumor stroma, wherein the antibody is operatively attached or conjugatedto at least a first cytotoxic or anti-cellular agent or to, e.g., atleast a first coagulation factor.

By way of example only, the targeted construct or conjugate may be anantibody construct or conjugate that specifically binds to a tumor cellsurface molecule; to a component of tumor vasculature, such asE-selectin, P-selectin, VCAM-1, ICAM-1, endoglin or an integrin; to acomponent adsorbed or localized in the vasculature or stroma, such asVEGF, FGF or TGFβ; to a component the expression of which is naturallyor artificially induced in the tumor environment, such as E-selectin,P-selectin or an MHC Class II antigen. Non-antibody targeting agentsinclude growth factors, such as VEGF and FGF; peptides containing thetripeptide R-G-D, that bind specifically to the tumor vasculature, andother targeting components such as annexins and related ligands.

The antibody constructs and conjugates may be operatively attached to atleast a first cytotoxic or otherwise anti-cellular agent. They may alsobe operatively attached to at least a first coagulation factor. Inattachment to coagulants, bispecific constructs may also beadvantageously employed (e.g., using two antibody binding regions),although the covalent linkages are generally preferred for use with thetoxins. Any one or more of the toxic or coagulating agents known in theart may be employed in such "immunotoxins" or "coaguligands", and TissueFactor or Tissue Factor derivatives may also be employed as part of thecoaguligands, where the coaguligand is the second, "anti-cancer agent".

The present invention therefore further provides methods for treating ananimal or patient having a vascularized tumor, which methods generallycomprise systemically administering to an animal one or morecoagulation-deficient Tissue Factor compounds and one or moreanti-cancer agents in a combined amount effective to coagulate the tumorvasculature and specifically induce tumor necrosis. The anti-canceragent may be a chemotherapeutic agent, as exemplified by etoposide, anantibody, or an antibody construct or conjugate comprising an antibodythat specifically binds to a component of a tumor cell, tumorvasculature or tumor stroma operatively attached to a cytotoxic agent orto a coagulation factor.

Whether the anti-cancer agent is a chemotherapeutic or antibody-basedconstruct, the one or more anti-cancer agent(s) may be administered tothe animal simultaneously, e.g., from a single composition or from twoor more distinct compositions. The staggered or sequentialadministration of the one or more Tissue Factor compounds and the one ormore anti-cancer agent(s) is also contemplated. The "sequentialadministration" requires that the TF and anti-cancer agent beadministered to the animal at "biologically effective time intervals".For example, the Tissue Factor compound(s) may be administered to theanimal at a biologically effective time prior to the anti-canceragent(s), or the anti-cancer agent(s) may be administered to the animalat a biologically effective time prior to the Tissue Factor compound(s).Where a Tissue Factor compound is administered first, it will generallybe given at a biologically effective time sufficient to allow the TissueFactor compound to preferentially localize within the tumor vasculatureprior to the administration of the anti-cancer agent(s).

The present invention further includes methods of using at least one ofFactor VIIa or an activator of Factor VII to increase the effectivenessof any one or more of the coagulation-deficient Tissue Factor (TF)compounds that define the primary therapeutic. Such methods generallycomprise further administering to an animal or patient a therapeuticallyeffective amount of Factor VIIa or an activator of Factor VII.

In such embodiments, the use of Factor VIIa itself will be generallypreferred. The Factor VIIa employed may consist essentially of the aminoacid sequence from amino acid 61 to amino acid 212 of the Factor VIIpolypeptide sequence of SEQ ID NO:14.

Again, the Factor VIIa or Factor VII activator may be administered tothe animal simultaneously with the coagulation-deficient Tissue Factorcompound. As such, Factor VIIa may be administered to the animal orpatient in a pre-formed Tissue Factor-Factor VIIa complex. In certainembodiments, the Tissue Factor-Factor VIIa complex will be an equimolarcomplex.

Further, the coagulation-deficient Tissue Factor compound and FactorVIIa compound may be administered to the animal using staggered orsequential administration. The prior administration of the Tissue Factorcompound will generally be preferred and it will preferably beadministered to the animal at a biologically effective time prior to theFactor VIIa compound. Such an effective prior administration of theTissue Factor compound will generally be at a biologically effectivetime sufficient to allow the Tissue Factor compound to preferentiallylocalize within the tumor vasculature prior to the administration of theFactor VIIa compound.

These methods of the invention may thus be further described as methodsfor promoting coagulation in the tumor vasculature of an animal orpatient having a vascularized tumor, comprising systemically providingto the animal or patient a coagulation-deficient Tissue Factor compoundand Factor VIIa or an activator of Factor VII in a combined amountsufficient to preferentially or specifically promote coagulation in thetumor vasculature.

The subject animal will preferably be provided with thecoagulation-deficient Tissue Factor compound at a time prior to theprovision of the Factor VIIa, wherein the time interval prior to FactorVIIa administration is effective for the Tissue Factor compound topreferentially or specifically localize within the tumor vasculature.

Further methods are described as methods for treating an animal having avascularized tumor, comprising systemically administering to the animala coagulation-deficient Tissue Factor compound and Factor VIIa in acombined amount effective to promote coagulation in the tumorvasculature and to specifically cause necrosis in the tumor. Thepre-administration of Tissue Factor is generally preferred such that theTissue Factor compound preferentially localize within the tumorvasculature and form a reservoir for subsequent Factor VIIa combination.

All such Factor VIIa combined treatments may be used with anycoagulation-deficient Tissue Factor compound, such as truncated,dimeric, and/or mutant Tissue Factors and/or those with increased halflives. These methods are particularly useful for combination with TissueFactor compounds that are deficient in the ability to activate FactorVII.

The combined treatment methods of the invention also encompass triplecombinations using one or more coagulation-deficient Tissue Factorcompounds, one or more anti-cancer agents and Factor VIIa or anactivator of Factor VII.

The present invention further provides novel compositions in the form ofcompositions that comprise one or more coagulation-deficient TissueFactor compounds that have been modified to increase their half life,other than wherein the modification consists of attaching the TissueFactor compound to an antibody that binds to a component of a tumorcell, tumor vasculature or tumor stroma.

The "increased half life Tissue Factor compounds" encompass all thecoagulation-deficient Tissue Factor compounds described above, such astruncated, dimeric, polymeric, and/or mutant Tissue Factors.

The increased half life Tissue Factor compounds preferably comprise acoagulation-deficient Tissue Factor compound that is operativelyattached, e.g., covalently attached, to a carrier molecule. Proteincarriers are currently preferred, as exemplified by albumins orglobulins, although non-protein carriers are also contemplated.

One class of increased half life coagulation-deficient Tissue Factorcompounds are those that are operatively attached to an antibody orportion thereof, such as an IgG molecule or to an Fc portion of anantibody. Tissue Factors introduced into a contiguous portion of an IgGmolecule, e.g., in place of the C_(H) 3 domain, are also contemplated.

The invention still further provides a series of novel therapeutic kitsfor use in conjunction with the methods of the invention. Certain kitswill comprise, preferably in suitable container means, at least a firstcoagulation-deficient Tissue Factor compound in combination with atleast a first anti-cancer agent.

The coagulation-deficient Tissue Factor compounds may be one or more ofthe coagulation-deficient Tissue Factors described herein, such astruncated, dimeric, polymeric, and/or mutant Tissue Factors, includingmutant Tissue Factors deficient in the ability to activate Factor VII.Where such Factor VII activation mutants are employed in the kit, thekit may optionally further comprise a biologically effective amount ofFactor VIIa.

The term "anti-cancer agent" is used as described above and coverschemotherapeutic agents, such as etoposide; and antibody-basedanti-cancer agents, such as antibody conjugates comprising an antibodythat specifically binds to a component of a tumor cell, tumorvasculature or tumor stroma operatively attached to a cytotoxic agent orto a coagulation factor, including a Tissue Factor or a Tissue Factorderivative.

Further therapeutic kits of the invention generally comprise, preferablyin suitable container means, a mutant Tissue Factor compound that isdeficient in the ability to activate Factor VII in combination withFactor VIIa. Previously, the mutants of this category have been thoughtto be so lacking in activity that they could not be used therapeuticallyto induce coagulation, but only to act as an antagonist of wild type TFand to inhibit coagulation. Only the combination of substantially activetruncated Tissue Factor with Factor VIIa has been previously proposed,this being in connection with the treatment of bleeding disorders.

The present invention thus provides the novel combination of a mutantTissue Factor compound that is more significantly impaired in itscoagulating ability than truncated Tissue Factor, preferably by virtueof being deficient in the ability to activate Factor VII, in conjunctionwith Factor VIIa. The Factor VIIa will become "exogenous Factor VIIa"following administration to an animal. These kits therefore preferablycomprise, in suitable container means, a biologically effective amountof a mutant Tissue Factor compound deficient in the ability to activateFactor VII in combination with a biologically effective amount of atleast one of Factor VIIa or an activator of Factor VII. Activators ofFactor VII may substitute for the Factor VIIa in such kits, or may beemployed in addition to the Factor VIIa. Supplementary agents may alsobe added.

The TF mutants for use in such kits are exemplified by those thatinclude a mutation in the amino acid region between about position 157and about position 167 of SEQ ID NO:1. These are exemplified by thosemutants that wherein, within SEQ ID NO:1, Trp at position 158 is changedto Arg; wherein Ser at position 162 is changed to Ala; wherein Gly atposition 164 is changed to Ala; or wherein Trp at position 158 ischanged to Arg and Ser at position 162 is changed to Ala. Further:examples are those mutant TFs that consist essentially of the aminoacid sequence of SEQ ID NO:8 or SEQ ID NO:9.

Combined treatment kits comprising, preferably in suitable containermeans, at least a first coagulation-deficient Tissue Factor compound, atleast a first anti-cancer agent and Factor VIIa or an activator ofFactor VII are also provided by the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1: Induction of coagulation of plasma by full length TF. Blood (A)is shown in contact with the cell membrane (B).

FIG. 2: Domain structure of TF. Depicted are the extracellular domain(A; amino acids 1-219), and the cell membrane (B). The NH₂ domain (10)is depicted as cross-hatched, the Factor VII/VIIa binding region (20) isdepicted as hatched. The transmembrane domain (40) begins at amino acid220 (30) and spans the cell membrane. The transmembrane domain of TF isdeleted or otherwise rendered non-functional to generate a functionaltTF of the present invention. In certain tTF compositions the NH₂ domainmay also be deleted or rendered non-functional.

FIG. 3: Model for tTF induced coagulation of tumor vasculature. Blood(A) is depicted in contact with the cell membrane (B) of the tumorvascular endothelium (C). The prothrombotic tumor endothelium has FactorIX, X (shown) or TFPI/Xa plus phosphatidyl serine (PS⁻) on its surface,which binds tTF-VII or tTF-VIIa, leading to coagulation.

FIG. 4A and FIG. 4B. FIG. 4A: Induction of coagulation by cell-boundtTF. A20 cells (10⁵ cells, 100 μl) were incubated with antibodies (0.33μg) and tTF (0.17 μg) for 1 hour at 4° C. Calcium chloride (12.5 mM) andcitrated mouse plasma were added to the cells and the time for the firstfibrin strands to form was recorded (clotting time, seconds; horizontalaxis). Sample number is shown on the vertical axis. Sample 1 includes noadded antibodies or tTF (control), sample 2 includes B21-2/10H10antibody, sample 3 includes tTF, sample 4 includes B21-2/OX7 antibodyplus tTF, sample 5 includes CAMPATH-2/10H10 antibody plus tTF, sample 6includes 10H10 F(ab')₂ antibody plus tTF, sample 7 includes 10H10 Fab'antibody plus tTF, sample includes B21-2 F(ab')₂ antibody plus tTF,sample 9 includes B21-2 Fab' antibody plus tTF, sample 10 includesB21-2/10H10 antibody plus tTF. FIG. 4B: Relationship between the numberof bound tTF molecules and plasma coagulation time. The A20 cells (10⁵cells, 100 μl) were incubated with varying concentrations of B21-2/10H10plus an excess of tTF for 1 hour at 4° C. in the presence of sodiumazide and were then washed, warmed to 37° C. Calcium chloride (12.5 mM)and citrated mouse plasma (a different batch from that in A) were addedto the cells and the time for the first fibrin strands to form wasrecorded (clotting time, seconds; vertical axis). The number of tTFmolecules bound to the cells (◯) was determined in a parallel study with¹²⁵ I-tTF (log scale; horizontal axis). Values represent the means ofthree measurements, with SD.

FIG. 5: Coagulation of mouse plasma by cell-associated tTF₂₁₉, H₆-N'-cys-tTF₂₁₉ and H₆ -tTF₂₁₉ -cys-C'. A20 lymphoma cells (I-A^(d)positive) were treated at room temperature with the "capture" bispecificantibody, B21-2/10H10, recognizing both I-A^(d) and tTF. Cells werewashed and two different preparations of tTF₂₁₉ [standard tTF₂₁₉ (◯) andtTF₂₁₉ (▴),], H₆ -N'-cys-tTF₂₁₉ (□) or H₆ -tTF₂₁₉ -cys-C' () were addedat a range of tTF concentrations (concentration, M; horizontal axis).Cells were washed and warmed to 37° C. Calcium and citrated mouse plasmawere added and the time for the first strands of fibrin to form wasrecorded (clotting time, seconds; vertical axis).

FIG. 6: Coagulation of mouse plasma by cell-associated H₆ -tTF₂₂₀-cys-C' and tTF₂₂₀ -cys-C'. A20 lymphoma cells (I-A^(d) positive) weretreated at room temperature with the "capture" bispecific antibody,B21-2/10H10, recognizing both I-A^(d) and tTF. Cells were washed andstandard tTF₂₁₉ (▪), H₆ -tTF₂₂₀ -cys-C' (◯) and tTF₂₂₀ -cys-C' () wereadded at a range of concentrations (concentration, M; horizontal axis).Cells were washed and warmed to 37° C. Calcium and citrated mouse plasmawere added and the time for the first strands of fibrin to form wasrecorded (clotting time, seconds; vertical axis).

FIG. 7: Coagulation of mouse plasma by cell associated H₆ -tTF₂₂₁-cys-C', tTF₂₂₁ -cys-C' and H₆ -tTF₂₂₁ -cys-C' dimer. A20 lymphoma cells(I-A^(d) positive) were treated at room temperature with the "capture"bispecific antibody, B21-2/10H10, recognizing both I-A^(d) and tTF.Cells were washed and standard tTF₂₁₉ (◯), H₆ - tTF₂₂₁ -cys-C' (◯),tTF₂₂₁ -cys-C' (□), or H₆ -tTF₂₂₁ -cys-C' dimer (▴) were added at arange of concentrations (concentration, M; horizontal axis). Cells werewashed and warmed to 37° C. Calcium and citrated mouse plasma were addedand the time for the first strands of fibrin to form was recorded(clotting time, seconds; vertical axis).

FIG. 8: Coagulation of mouse plasma by cell-associated H₆ -N'-cys-tTF₂₁₉and H₆ -N'-cys-tTF₂₁₉ dimer. A20 lymphoma cells (I-A^(d) positive) weretreated at room temperature with the "capture" bispecific antibody,B21-2/10H10, recognizing both I-A^(d) and tTF. Cells were washed andstandard tTF₂₁₉ (◯), H₆ -N'-cys-tTF₂₁₉ (▪) and H₆ -N'-cys-tTF₂₁₉ dimer() were added at a range of concentrations (concentration, M;horizontal axis). Cells were washed and warmed to 37° C. Calcium andcitrated mouse plasma were added and the time for the first strands offibrin to form was recorded (clotting time, seconds; vertical axis).

FIG. 9: Thrombosis of vessels in large C1300 Muγ tumors by tTF₂₁₉. Nu/Numice bearing large (>1000 mm³) subcutaneous C1300 Muγ tumors wereinjected intravenously with 16-20 μg tTF₂₁₉. Twenty-four hours later,mice were anesthetized, exsanguinated and tumors and organs wereremoved. Paraffin sections of the tissues were evaluated for thepresence of thrombosed vessels. The numbers of thrombosed vessels andopen vessels in sections of tumors were counted. The percent of tumorvessels thrombosed is shown on the vertical axis. The hatched barrepresents tTF₂₁₉ injected mice, the open bar represents PBS injectedmice.

FIG. 10: Thrombosis of vessels in large C1300 tumors by tTF₂₁₉. Nu/numice bearing large (>1000 mm³) subcutaneous C1300 tumors were injectedintravenously with 16-20 μg tTF₂₁₉. Twenty-four hours later, mice wereanesthetized, exsanguinated and tumors and organs were removed. Paraffinsections of the tissues were evaluated for the presence of thrombosedvessels. The numbers of thrombosed vessels and open vessels in sectionsof tumors were counted. The percent of tumor vessels thrombosed is shownon the vertical axis. The hatched bar represents tTF₂₁₉ injected mice,the open bar represents PBS injected mice.

FIG. 11: Thrombosis of vessels in large 3LL tumors by tTF₂₁₉. C57BL/6mice bearing large (>800 mm³) subcutaneous 3LL tumors were injectedintravenously with 16-20 μg tTF₂₁₉. Twenty-four hours later, mice wereanesthetized, exsanguinated and tumors and organs were removed. Paraffinsections of the tissues were evaluated for the presence of thrombosedvessels. The numbers of thrombosed vessels and open vessels in sectionsof tumors were counted. The percent of tumor vessels thrombosed is shownon the vertical axis. The hatched bar represents tTF₂₁₉ injected mice,the open bar represents PBS injected mice.

FIG. 12A and FIG. 12B: Inhibition of growth of C1300 Muγ tumors in miceby tTF₂₁₉. FIG. 12A: Mice with 0.8 to 1.0 cm diameter C1300(Muγ) tumorswere given two intravenous injections of B21-2/10H10-tTF coaguligand ()spaced 6 days apart (arrows). Mice in control groups received equivalentdoses of tTF alone (□), CAMPATH-2/10H10 plus tTF (Δ), or phosphatebuffered saline (◯). Mice that received B21-2/OX7 and tTF had similartumor responses to those in animals receiving tTF alone. Administrationof B21-2/10H10 alone did not affect tumor growth. Each group contained12 to 27 mice. Points represent the mean tumor volume per group (±SEM).Mean tumor volume (cm³) is shown on the vertical axis, days after firsttreatment is shown on the horizontal axis. FIG. 12B: Nu/nu mice bearingsmall (350 mm³) subcutaneous C1300 Muγ tumors were injectedintravenously with 16-20 μg tTF₂₁₉ (▪) or phosphate buffered saline (◯).The treatment was repeated one week later. Tumors were measured dailyand tumor volumes (+one standard deviation) were calculated. The numberof mice per treatment group was 8-10. Mean tumor volume (cm³) is shownon the vertical axis, days after first treatment is shown on thehorizontal axis.

FIG. 13: Inhibition of growth of H460 tumors in mice by tTF₂₁₉. Nu/numice bearing small (350 mm³) subcutaneous H460 tumors were injectedintravenously with 16-20 μg tTF₂₁₉ (▪) or PBS (◯). The treatment wasrepeated one week later. The time of injections are designated byarrows. Tumors were measured daily and tumor volumes (+one standarddeviation) were calculated. The number of mice per treatment group was8-10. Mean tumor volume (cm³) is shown on the vertical axis, days afterfirst treatment is shown on the horizontal axis.

FIG. 14: Inhibition of growth of HT29 tumors in mice by tTF₂₁₉. Nu/numice bearing large (1200 mm³) subcutaneous HT29 tumors were injectedintravenously with 16 μg or 64 μg tTF₂₁₉ (□) or PBS (). Tumors weremeasured daily (days after injection; horizontal axis), and tumorvolumes (+one standard deviation) were calculated (tumor volume, mm³ ;vertical axis). The number of mice per treatment group was 3-4.

FIG. 15: Coagulation of mouse plasma by cell-associated IgG-H₆-N'-cys-tTF₂₁₉. A20 lymphoma cells (I-A^(d) positive) were treated withthe "capture" bispecific antibody, B21-2/10H10, recognizing both I-A^(d)and tTF₂₁₉. IgG-H₆ -N'-cys-tTF₂₁₉ (Δ), H₆ -N'-cys-tTF₂₁₉ (▪) or tTF₂₁₉(◯) were added at a range of concentrations at room temperature(concentration, M; horizontal axis). Cells were washed and warmed to 37°C. Calcium and citrated mouse plasma were added and the time for thefirst strands of fibrin to form was recorded (clotting time, seconds;vertical axis).

FIG. 16: Coagulation of mouse plasma by cell-associated IgG-H₆-N'-cys-tTF₂₁₉ and IgG-H₆ -tTF₂₁₉ -cys-C'. Immunoglobulin-tTF conjugateswere prepared by linking B21-2 IgG (against I-A^(d)) to H₆-N'-cys-tTF₂₁₉ (▴) or H₆ -tTF₂₁₉ -cys-C' (▪). The conjugates were addedat a range of concentrations to A20 lymphoma cells (I-A^(d) positive) atroom temperature, and compared to tTF₂₁₉ () (concentration, M;horizontal axis). Cells were washed and warmed to 37° C. Calcium andcitrated mouse plasma were added. The time (seconds) for the firststrands of fibrin to form was recorded. The vertical axis shows clottingtime as a percent of the control.

FIG. 17: Conversion of Factor X to Factor Xa by cell-associated IgG-H₆-N'-cys-tTF₂₁₉ and Fab'-H₆ -N'-cys-tTF₂₁₉, measured by a chromogenassay. A20 cells (I-A^(d) positive) were treated with the "capture"bispecific antibody, B21-2/10H10, recognizing both I-A^(d) and tTF, wasadded with IgG-H₆ -N'-cys-tTF₂₁₉ (◯) or Fab'-H₆ -N'-cys-tTF₂₁₉ (Δ),which were added at a range of concentrations at room temperature.B21-2/10H10 plus H₆ -N'-cys-tTF₂₁₉ (×) and Mac51/10H10 plus H₆-N'-cys-tTF₂₁₉ (control, ▪) were also added (concentration, M;horizontal axis). Cells were washed and warmed to 37° C. Calcium and"Proplex T" were added (Proplex T contains Factors II, VII, IX and X).The production of Xa was measured by adding the chromophore-releasingsubstrate, S-2765, and measuring the optical density at 409 nm (OD₄₀₉nm; vertical axis).

FIG. 18: Inhibition of growth of C1300 Muγ tumors in mice byimmunoglobulin-tTF conjugate. Nu/nu mice bearing small (300 mm³)subcutaneous C1300 Muγ tumors were injected intravenously with 16-20 μgtTF₂₁₉ complexed with OX7 Fab'/10H10 Fab bispecific "carrier" antibody(Δ). Other mice received tTF₂₁₉ alone (▪), or diluent (PBS, ◯). Thetreatment was repeated one week later. The day treatments were given aredesignated by arrows. Tumors were measured daily and tumor volumes (+onestandard deviation) were calculated. The number of mice per treatmentgroup was 7-10. Mean tumor volume (cm³) is shown on the vertical axis,days after first treatment is shown on the horizontal axis.

FIG. 19: Enhancement of anti-tumor activity of immunoglobulin-tTF byetoposide. SCID mice bearing subcutaneous L540 human Hodgkin's tumorswere given a single intravenous injection of a complex of tTF₂₁₉ and the"carrier" bispecific antibody Mac51Fab'/10H10 Fab' (▪). Other micereceived 480 μg of etoposide intraperitoneally 2 days before, 1 daybefore and on the day of treatment with immunoglobulin-tTF conjugate(▴). Other mice received etoposide alone (◯) or diluent (PBS, Δ). Tumorswere measured daily and tumor volumes (+one standard deviation) werecalculated. Mean tumor volume (cm³) is shown on the vertical axis, daysafter treatment is shown on the horizontal axis.

FIG. 20: Enhancement of plasma coagulation by Factor VIIa. A20 lymphomacells (I-A^(d) positive) were treated at room temperature with the"capture" bispecific antibody, B21-2/10H10, recognizing both I-A^(d) andtTF. Cells were washed and tTF₂₁₉ alone (◯) or tTF₂₁₉ with Factor VIIawere added at a range of concentrations of Factor VIIa, as follows: 0.1nM (▪); 0.3 nM (); 0.9 nM (Δ); 2.7 nM (▴); and 13.5 nM (+)(concentration, M; horizontal axis). Cells were washed and warmed to 37°C. Calcium and citrated mouse plasma were added and the time for thefirst strands of fibrin to form was recorded (clotting time, seconds;vertical axis).

FIG. 21: Weak coagulation of mouse plasma by cell associated tTF₂₁₉(W158R) and tTF₂₁₉ (G164A) mutants. A20 lymphoma cells (I-A^(d)positive) were treated at room temperature with the "capture" bispecificantibody, B21-2/10H10, recognizing both I-A^(d) and tTF. Cells werewashed and tTF₂₁₉ (◯), tTF₂₁₉ (W158R) () or tTF₂₁₉ (G164A) (□) wereadded at a range of concentrations (concentration, M; horizontal axis).Cells were washed and warmed to 37° C. Calcium and citrated mouse plasmawere added and the time for the first strands of fibrin to form wasrecorded (clotting time, seconds; vertical axis).

FIG. 22: Restoration of the coagulation-inducing activity of mutanttTF₂₁₉ (G164A) and (W158R) by Factor VIIa. A20 lymphoma cells (I-A^(d)positive) were treated at room temperature with the "capture" bispecificantibody, B21-2/10H10, recognizing both I-A^(d) and tTF. Cells werewashed and not treated (Δ), or were treated with: tTF₂₁₉ (◯); tTF₂₁₉(G164A) (▪) or tTF₂₁₉ (W158R) (); each with Factor VIIa addition at arange of concentrations (concentration, nM; horizontal axis). Cells werewashed and warmed to 37° C. Calcium and citrated mouse plasma were addedand the time for the first strands of fibrin to form was recorded(clotting time, seconds; vertical axis).

FIG. 23: Antitumor activity of tTF219:VIIa and tTF219 (G164A):VIIacomplexes in mice bearing HT29 human colorectal carcinomas. From left toright, the bars represent: saline (1); tTF (2); Factor VIIa (3); tTFplus Factor VIIa (4); G164A (5); and G164A plus Factor VIIa (6). Thevertical axis shows the average percent of necrosis in tumors examined.

SEQUENCE SUMMARY

    ______________________________________                                        SEQ ID NO: 1                                                                            Amino Acid Sequence of tTF.sub.219                                  SEQ ID NO: 2                                                                            Amino Acid Sequence of H.sub.6 -N'-cys-tTF.sub.219                  SEQ ID NO: 3                                                                            Amino Acid Sequence of H.sub.6 -tTF.sub.219 -cys-C'                 SEQ ID NO: 4                                                                            Amino Acid Sequence of N'-cys-tTF.sub.219                           SEQ ID NO: 5                                                                            Amino Acid Sequence of tTF.sub.219 -cys-C'                          SEQ ID NO: 6                                                                            Amino Acid Sequence of H.sub.6 -tTF.sub.220 -cys-C'                 SEQ ID NO: 7                                                                            Amino Acid Sequence of H.sub.6 -tTF.sub.221 -cys-C'                 SEQ ID NO: 8                                                                            Amino Acid Sequence of tTF.sub.219 (W 158 R)                        SEQ ID NO: 9                                                                            Amino Acid Sequence of tTF.sub.219 (G 164 A)                        SEQ ID NO: 10                                                                           cDNA sequence for tTF.                                              SEQ ID NO: 11                                                                           Full genomic sequence of Tissue Factor                              SEQ ID NO: 12                                                                           Amino acid sequence of Tissue Factor                                SEQ ID NO: 13                                                                           Factor VII DNA                                                      SEQ ID NO: 14                                                                           Factor VII amino acid                                               SEQ ID NO: 15                                                                           5' primer for tTF amplification                                     SEQ ID NO: 16                                                                           3' Primer for tTF amplification                                     SEQ ID NO: 17                                                                           5' primer GlytTF complimentary DNA amplification                              primer                                                              SEQ ID NO: 18                                                                           5' primer for Preparation of tTF and the 5' half of the                       linker DNA                                                          SEQ ID NO: 19                                                                           3' primer for Preparation of tTF and the 5' half of the                       linker DNA                                                          SEQ ID NO: 20                                                                           5' primer for Preparation of the 3' half of the linker                        DNA and tTF DNA                                                     SEQ ID NO: 21                                                                           3' primer for Preparation of the 3' half of the linker                        DNA and tTF DNA                                                     SEQ ID NO: 22                                                                           5' primer for Cys [tTF] Linker [tTF] construction                   SEQ ID NO: 23                                                                           3' primer for Cys [tTF] Linker [tTF] construction                   SEQ ID NO: 24                                                                           5' primer for [tTF] Linker [tTF]cys                                 SEQ ID NO: 25                                                                           3' primer for [tTF] Linker [tTF]cys                                 SEQ ID NO: 26                                                                           primer for [tTF] G164A formation                                    SEQ ID NO: 27                                                                           primer for [tTF] W158R S162A                                        ______________________________________                                    

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Solid tumors and carcinoma account for more than 90% of all cancers inman (Shockley et al., 1991). The therapeutic uses of monoclonalantibodies and immunotoxins have been investigated in the therapy oflymphomas and leukemias (Lowder et al., 1987; Vitetta et al., 1991), buthave been disappointingly ineffective in clinical trials againstcarcinomas and other solid tumors (Byers and Baldwin, 1988; Abrams andOldham, 1985).

A principal reason for the ineffectiveness of antibody-based treatmentsis that macromolecules are not readily transported into solid tumors(Sands, 1988; Epenetos et al., 1986). Even when these molecules get intothe tumor mass, they fail to distribute evenly due to the presence oftight junctions between tumor cells (Dvorak et al., 1991), fibrousstroma (Baxter et al., 1991), interstitial pressure gradients (Jain,1990) and binding site barriers (Juweid et al., 1992).

In developing new strategies for treating solid tumors, the methods thatinvolve targeting the vasculature of the tumor, rather than the tumorcells themselves, offer distinct advantages. Inducing a blockade of theblood flow through the tumor, e.g., through tumor vasculature specificfibrin formation, would interfere with the influx and efflux processesin a tumor site, thus resulting in anti-tumor effect. Arresting theblood supply to a tumor may be accomplished through shifting theprocoagulant-fibrinolytic balance in the tumor-associated vessels infavor of the coagulating processes by specific exposure to coagulatingagents. Accordingly, antibody-coagulant constructs and bispecificantibodies have been generated and used in the specific delivery of acoagulant to the tumor environment (WO 96/01653).

However, the requirement for specificity, although not so stringent aswith immunotoxins, is still important. To achieve specificity, it hasgenerally been believed that an effector molecule, whether a toxin or acoagulant, needs to be conjugated or functionally associated with atargeting molecule, such as an antibody or other ligand with specificityfor the tumor environment. Such targeting entities may be directed tothe tumor cells themselves, although it is now believed to be preferableto use targeting molecules directed against components of the tumorvasculature or tumor stroma. A number of appropriate target moleculeshave been identified that are specifically or preferentially expressed,localized, adsorbed to or inducible on the cells or in the environmentof the tumor vasculature and/or stroma.

Although the tumor vasculature and stroma targeting methods can be quiteeffective, it will be recognized that to practice such targetingmethodology still requires a certain knowledge and requires thepreparation of suitable conjugates or coordinated molecular complexes.For example, in targeting a coagulant to the tumor vasculature, one mustidentify an appropriate vascular antigen, prepare an antibody or ligandthat binds to the target antigen, choose an appropriate coagulant, linkthe coagulant to the antibody or ligand or otherwise form a functionalassociation of the two components, and conduct the localizationprotocols using doses that do not result in significant mis-direction ofthe agent. Although such methods can be readily and successfullypracticed, one can see that advantages would result from the developmentof methodology that included less preparative steps and could thereforebe performed in a more cost-effective manner.

The present invention provides such new methods for effecting specificblood coagulation, as exemplified by tumor-specific coagulation, withoutthe need for targeting molecules, such as antibodies. This is achievedby administering compositions comprising coagulant-deficient TissueFactor, which was discovered to specifically promote coagulation in thetumor vasculature, despite the fact that it lacks any recognized tumortargeting component. The present invention provides that suchcoagulation-impaired TF compositions may be administered alone, as TFconjugates with improved half-life, in combination with conventionalchemotherapeutics, in combination with targeted immunotoxins orcoaguligands, in combination with Factor VIIa (FVIIa) or FVIIaactivators or in any of the foregoing combinations.

A. Tissue Factor

Tissue Factor (TF) is the major initiating receptor for the thrombogenic(blood coagulation) cascades (Davie, et al. 1991). TF is a single chain,263 amino acid membrane glycoprotein (SEQ ID NO:12), and its primarysequence has structural similarity with the chemokine receptor family(Edgington et al., 1991). TF is a transmembrane cell surface receptorand functions as the receptor and cofactor for Factor VIIa. TF bindsFactor VIIa to form a proteolytically active complex on the cell surface(Ruf and Edgington, 1991b, 1994; Ruf et al., 1991, 1992a, 1992b). Thiscomplex rapidly activates the serine protease zymogens Factors IX and Xby limited proteolysis, leading to the formation of thrombin and,ultimately, a blood clot (FIG. 21).

Thus, TF is an activator of the extrinsic pathway of blood coagulationand is not in direct contact with the blood under physiologically normalconditions (Osterud et al., 1986; Nemerson, 1988; Broze, 1992; Ruf andEdgington, 1994). In vascular damage or activation by certain cytokinesor endotoxin, however, TF will be exposed to the blood, either by the(sub)endothelial cells (Weiss et al., 1989) or by certain blood cells(Warr et al., 1990). TF will then complex with Factor VIIa, which undernormal conditions circulates at low concentrations in the blood(Wildgoose et al., 1992), and the TF/Factor VIIa complex will start thecoagulation cascade through the activation of factor X into Factor Xa.The cascade will ultimately result in the formation of fibrin (FIG. 1).For this sequence of events to occur, the TF:VIIa complex has to beassociated with a phospholipid surface upon which thecoagulation-initiation complexes with Factors IX or X can assemble (Rufand Edgington, 1991a; Ruf et al., 1992c; Paborsky et al., 1991; Bach etal., 1986; Krishnaswamy et al., 1992; ten Cate et al., 1993).

A limited number of cells constitutively express TEF. Lung and centralnervous system tissues contain high levels of TF activity, with TF beingfound in bronchial mucosa and alveolar epithelial cells in the lung andin glial cells and astrocytes in the nervous system. Expression of TFhas also been reported in cardiac myocytes, renal glomeruli, and incertain epithelial or mucosal tissues of the intestine, bladder andrespiratory tract. It can thus be seen that TF is generallyconstitutively expressed at tissue barriers between body tissues and theexternal environment (Drake et al., 1989; Ruf and Edgington, 1994).

TF is also present at tissue boundaries between organs, such as in theorgan capsules of the liver, spleen and kidney, and is also present inthe adventitia of arteries and venules. The expression of TF in thismanner allows TF to function in the arrest of internal bleeding. It istherefore relevant to note that TF is absent in the joints and skeletalmuscle of hemophiliacs, which are the primary sites of bleeding in thesepatients.

TF is typically not expressed to any significant degree on cells of theblood or the surface of endothelial cells that form the vasculatureunder normal conditions, but its expression by (sub)endothelial cellsand monocytes within the vasculature can be induced by infectiousagents. Monocytes, for example, are induced to express TF by cytokinesand T cells. Expression of TF in the vasculature typically will resultin disseminated intravascular coagulation or localized initiation ofblood clots or thrombogenesis. In this context, it is important to notethat TF must be available at all sites of the body where coagulationwould be necessary following tissue damage, infection or other insults.Therefore, TF should be equally available to all such tissue sites andshould not be generally reserved within any particular localized area ofthe body.

Certain studies have led to the delineation of a connection between TFand the development of the neoplastic phenotype in certain types oftumors (Ruff and Edgington, 1994). In fact, increasing levels of TF havebeen reported to be a prognostic indicator of the metastatic potentialof malignant melanoma (Mueller, et al., 1992). It has been reasoned thata generalized activation of the coagulation cascade could damage thevasculature leading to access of tumor cells or tumor cell-derivedvesicles to the general circulation, allowing such tumor cells to seedand cause metastatic tumor outgrowth.

Irrespective of the underlying mechanism, the studies described abovehave led Edgington and colleagues to propose the use of antibodiesdirected against TF in cancer treatment (WO 94/05328). These authorshave therefore proposed that antibodies with binding affinity for TFhave therapeutic utility in cancer treatment, particularly in connectionwith those patients believed to be at risk for the development ofmetastatic tumors. This intent has led to the development of hybridomasproducing monoclonal antibodies that react with human TF (U.S. Pat. No.5,223,427).

In addition to the use in cancer treatment, anti-TF antibodies have alsobeen proposed for use in inhibiting excessive coagulation, which mayalso be used in connection with the treatment of septic shock and inmoderating inflammatory responses (Morrissey et al., 1988; U.S. Pat. No.5,223,427), or in the treatment of myocardial infarction, where theantibodies are used as TF antagonists (U.S. Pat. No. 5,589,173). Thecombined use of anti-TF antibodies and other thrombolytic agents todissolve occluding thrombi is particularly disclosed in U.S. Pat. No.5,589,173. A specific method for using such antibodies is in theinhibition of coagulation in an extracorporeal circulation procedure inwhich blood is removed from a patient during a surgical procedure, suchas a cardiopulmonary bypass procedure (U.S. Pat. No. 5,437,864).

As is developed more fully below (Section B), human TF has been clonedand available for some time (Morrissey et al., 1987; Edgington et al.,1991; U.S. Pat. No. 5,110,730). In certain early studies, the sameprotein currently identified as human TF may be referred to as human TFheavy chain protein or the heavy chain of TF. The gene encodes apolypeptide precursor of 295 amino acids in length, which includes apeptide leader with alternative cleavage sites, which is now known tolead to the formation of a protein of 263 amino acids in length. Therecombinant expression of human TF in CHO cells has been reported tolead to the production of TF at a level that is described as being oneof the highest expression levels reported for a recombinanttransmembrane receptor following production in mammalian cells(Rehemtulla et al., 1991).

A recombinant form of TF has been constructed that contains only thecell surface or extracellular domain (Stone, et al., 1995) and lacks thetransmembrane and cytoplasmic regions of TF. This `truncated` TF (tTF)is 219 amino acids in length and is a soluble protein with approximately10⁵ times less factor X-activating activity relative to nativetransmembrane TF in an appropriate phospholipid membrane environment(Ruf, et al., 1991b). This difference in activity is because the TF:VIIacomplex binds and activates Factors IX and X far more efficiently whenassociated with a negatively charged phospholipid surface (Ruf, et al,1991b; Paborsky, et al., 1991).

Despite the significant impairment of coagulative capacity of the tTF,tTF can promote blood coagulation when tethered or functionallyassociated by some other means with a phospholipid or membraneenvironment. For example, it is demonstrated herein that using abispecific antibody that binds tTF to a plasma membrane antigen allowsrestoration of useful coagulating activity. This led one of the presentinventors to develop methods for the specific coagulation of tumorvascular in vivo by using targeting constructs to deliver tTF orvariants thereof specifically to the tumor vascular or stroma (WO96/01653). Intravenous administration of such a "coaguligand" leads tolocalization of the coagulants within the tumor, thrombosis of the tumorvessels, and resultant tumor necrosis.

The development of the intelligent, targeted delivery of coagulants tothe tumor vasculature, as exemplified using a bispecific targetingantibody-tTF composition, may be seen as representing an improvementover classic immunotoxin therapy. In fact, such coaguligand treatmentinduces thrombosis of tumor vessels in less than 30 minutes, incomparison to about 6 hours necessary to achieve the same effectfollowing administration of an immunotoxin. Furthermore, there was nonotable side effects as a result of the coaguligand treatment. Althoughthe targeted delivery of a coagulant such as tTF was surprisinglyeffective, this stills requires the preparation of the "targetingconstruct".

Other studies of TF with vastly different objectives have also beenreported to identify uses for tTF that do not rely on their associationwith a targeting agent. In this regard, tTF has lately been consideredas a candidate for use in treating disorders such as hemophilia. Thiswork may have developed from the attempts to use apo-TF in suchtreatments. Apo-TF is a delipidated preparation of TF that was proposedfor infusion into hemophiliacs, based upon the hypothesis that thismolecule would spontaneously and preferentially incorporate itself orassociate with exposed membrane surfaces available at sites of injury.Thus, it was reasoned that apo-TF could be useful in such treatmentswithout leading to significant side effects (O'Brien et al., 1988; U.S.Pat. No. 5,017,556).

The apo-TF therapy has been proposed for use in chronic bleedingdisorders characterized by a tendency towards hemorrhage, both inheritedand acquired. U.S. Pat. No. 5,017,556 describes such disorders as thoseconnected with the deficiency of Factors VIII, IX or XI; or thoseconnected with the acquisition of inhibitors to Factors V, VIII, IX, XI,XII and XIII. The use of apo-TF, characterized as being substantiallydevoid of the naturally occurring lipid of Tissue Factor and possessingsubstantially no procoagulant activity prior to administration, wasacknowledged to be in contrast to the expected results, which would havebeen reasoned to lead to toxicity. It now appears that the resultsdescribed in U.S. Pat. No. 5,017,556 generally represent an anomaly inthe art, and these studies have been contradicted by other researchersworking in this field.

In fact, during attempts to put studies based upon those described aboveinto practice, experimental animals were observed to develop sideeffects such as disseminated intervascular coagulation (DIC). This ledto the conclusion that the intravenous administration of apo-TF is toodangerous to use (Sakai and Kisiel, 1990; U.S. Pat. Nos. 5,374,617;5,504,064; and 5,504,067).

The development of the soluble, truncated form of TF has not beenrecognized as solving the problems associated with TF or apo-TF. Forexample, tTF has been dismissed as an alternative to TF, due to the factthat it has been characterized as having almost no procoagulant activitywhen tested with normal plasma (Paborsky et al., 1991; U.S. Pat. No.5,374,617).

The potential uses for tTF possible prior to the present invention arethus confined to the targeted delivery of tTF, e.g., using antibodies,and the possible use of tTF to treat a limited number of disorders whenused in combination with other accessory molecules necessary forrestoration of sufficient activity (U.S. Pat. No. 5,374,617). Thissecond possibility has been exploited in certain limited circumstancesby combining the use of tTF with the administration of the clottingfactor, Factor VIIa. The combined use of Factor VIIa with tTF results inrestoration of sufficient coagulant activity for this combination to beof use in treating bleeding disorders, such as hemophilia. However, incontrast to the targeted delivery of coagulants such as tTF discussed inWO 96/01653, the tTF and Factor VIIa combination therapy includes noconcept of specific targeting. This therapy has therefore been proposedfor use only in connection with patients in which coagulation isimpaired (U.S. Pat. Nos. 5,374,617; 5,504,064; and 5,504,067).

The group of patients most readily identified with such impairedcoagulation mechanisms are hemophiliacs, including those suffering fromhemophilia A and hemophilia B, and those that have high titers ofantibodies directed to clotting factors. In addition, this combined tTFand Factor VIIa treatment has been proposed for use in connection withpatients suffering from severe trauma, post-operative bleeding or evencirrhosis (U.S. Pat. Nos. 5,374,617; 5,504,064; and 5,504,067). Bothsystemic administration by infusion and topical application have beenproposed as useful in such therapies. These therapies can thus be seenas supplementing the body with two clotting type "factors" in order toovercome any natural limitations in these or other related molecules inthe coagulation cascade in order to arrest bleeding at a specific site.

Roy et al. have also proposed the use of certain Tissue Factor mutantsin the treatment of myocardial infarction, particularly in theprevention of the reocclusion of coronary arteries (U.S. Pat. No.5,346,991). As such, the Tissue Factor mutants are being used as"thrombolytic agents", and are described as medicaments capable oflysing a fibrin-platelet thrombus in order to permit blood to again flowthrough an affected blood vessel. The TF mutants described are designedwith the intention of being capable of neutralizing the effects ofendogenous TF. Their use in connection with myocardial infarctiontherapy is said to permit early reperfusion, prevent reocclusion and totherefore limit tissue necrosis.

The artificial means of recreating the natural environment in thecontext described above is linked to the natural processes, whereinTissue Factor was described as being constitutively present atboundaries between organs in order to allow it to function as aninitiating molecule to arrest the bleeding. However, such limitation ofbleeding episodes in hemophiliacs naturally needs to be achieved withouttipping the balance of the coagulation pathways into widespreadcoagulation, which would be detrimental to such patients and wouldinhibit the oxygen supply to the particular tissue or organ in question.Therefore, widespread circulation and activity of tTF would beundesirable and would not, in fact, be expected to occur from thestudies described above.

Although tTF has not previously been shown to have any capacity topreferentially localize within a given site, and despite its knowngreatly diminished coagulative ability relative to native, full lengthTissue Factor, the present invention demonstrates that whensystematically administered to animals with solid tumors, tTF inducesspecific coagulation of the tumor's blood supply, resulting in tumorregression. The various aspects of the present invention are thereforebased on the discovery of the selective thrombosis of the tumor vesselsby tTF.

A1. Coagulation-Deficient TF

The surprising finding of the inventors that tTF specifically localizedwithin tumors sufficiently so as to cause anti-tumor effect wasdiscovered during studies using tTF as a control in antibody-coagulant("coaguligand") tumor targeting studies. From this initial discovery,the inventors developed the various aspects of the invention disclosedherein. The Tissue Factor compounds or constructs for use in the presentinvention have thus been developed from the original tTF first employed.Accordingly, various TF constructs may now be employed, including manydifferent forms of tTF, longer but still impaired TFs, mutants TFs, anytruncated, variant or mutant TFs modified or otherwise conjugated toimprove their half-life, and all such functional equivalents thereof.However, it will be understood that each of the TF constructs for use inthe invention are unified by the need to be "coagulation-deficient". Asdetailed herein below, there are various structural considerations thatmay be employed in the design of candidate coagulation-deficient TFs,and various assays are available for confirming that the candidate TFsare indeed suitable for use in the treatment aspects of the presentinvention. Given that the technological skills for creating a variety ofcompounds, e.g., using molecular biology, are routine to those ofordinary skill in the art, and given the extensive structural andfunctional guidance provided herein, the ordinary artisan will bereadily able to make and use a number of different coagulation-deficientTFs in the context of the present invention.

Also as described in significant detail herein, any one or more of thevariety of TFs may also be combined with other agents for use in theadvantageous treatment of solid tumors and other diseases associatedwith prothrombotic fluid vessels. In addition to combination withstandard treatments, such as surgery and radiotherapy, the coagulationapproach of the present invention may also be combined with theadministration of classical chemotherapeutic drugs, other immunotoxinsor coaguligands, or with additional clotting factors, as exemplified byFactor VIIa.

Given that the combined treatments of the invention are expected to givean additive, enhanced or even synergistic anti- tumor effect, those ofskill in the art will also readily appreciate that TF constructs thathave less than optimal properties in the types of in vitro and in vivoassays described herein may still be used in the context of the presentinvention. For example, should a candidate coagulation-deficient TFconstruct have a coagulating activity towards the lower end of the scalerecommended herein, such a molecule may still prove to be useful incombination with chemotherapeutics, clotting factors or otheranti-cancer agents. Equally, candidate coagulation-deficient TFconstructs that may be considered to have a coagulating activitysufficiently high to cause concerns regarding side effects, may stillprove to be useful after careful in vivo studies using experimentalanimals and in clinical studies beginning with low doses. Therefore, thefollowing guidelines concerning the coagulation-deficient TF moleculesare provided only as exemplary teaching, and those of ordinary skill inthe art will readily appreciate that TF molecules that do not exactlyfit within the structural and quantitative guidelines presented hereinmay still have significant therapeutic utility in the context of thepresent invention. Although determining this fact may often generallyrequire in vivo tests in animals, such tests are routine to those ofordinary skill in the art and simply require administration andmonitoring.

A2. Structural Considerations for Coagulation-Deficient TF

Those of skill in the art will readily appreciate that the TF moleculesfor use in the present invention cannot be substantially native TF. Thisis evident as natural TF and close variants thereof are particularlyactive in promoting coagulation. Therefore, upon administration to ananimal or patient, this would lead to widespread coagulation and wouldbe lethal. Therefore, formulations of intact, natural TF should beavoided. Likewise, attempts to modulate the TF activity by manipulatingits physical environment are not believed to be particularly productivein the context of the present invention. For example, the apo-TFapproach of O'Brien and colleagues (1988) should be avoided due to theDIC that is expected to result.

FIG. 2 is provided herein as an instructive model concerning the domainsof the native TF molecule. It is an objective of the invention toprovide TF molecules that do not substantially associate with the plasmamembrane. Naturally, truncation of the molecule is the most directmanner in which to achieve a modified TF that does not bind to themembrane. These types of truncated constructs are described more fullybelow. However, actual truncation or shortening of the molecule is notthe only mechanism by which operative TF variants may be created. By wayof example only, mutations may be introduced into the C-terminal regionof the molecule that normally traverses the membrane in order to preventproper membrane insertion. It is contemplated that the insertion ofvarious additional amino acids, or the mutation of those residuesalready present, may be used to effect such membrane expulsion.Therefore, modifications that may be considered in this regard are thosethat reduce the hydrophobicity of the C-terminal portion of the moleculeso that the thermodynamic properties of this region are no longerfavorable to membrane insertion.

In considering making structural modifications to the native TFmolecule, those of skill in the art will be aware of the need tomaintain significant portions of the molecule sufficient for theresultant TF variant to be able to function to promote at least somecoagulation. An important consideration is that the TF molecule shouldsubstantially retain its ability to bind to Factor VII or Factor VIIa.By reference to FIG. 2, it will be seen that the VII/VIIa binding regionis generally central to the molecule and such region should therefore besubstantially maintained in all TF variants proposed for use in thepresent invention. The particular location of this binding region andoptional use of mutants, either alone or in combination with otheragents, is discussed in more detail below.

Nonetheless, certain sequence portions from the N-terminal region of thenative TF are also contemplated to be dispensable. Therefore, one mayintroduce mutations into this region or may employ deletion mutants(N-terminal truncations) into the candidate TF molecules for useherewith. Given these guidelines, those of skill in the art willappreciate that the following exemplary truncated, dimeric, multimericand mutant TF constructs are by no means limiting and that many otherfunctionally equivalent molecules may be readily prepared and used.

A3. Exemplary Coagulation-Deficient TF Constructs

The following exemplary Tissue Factor compositions, including thetruncated, dimeric, multimeric and mutated versions, may exist asdistinct polypeptides or may be conjugated to inert carriers, such asimmunoglobulins, as described herein below.

i. Truncated Tissue Factor

As used herein, the term "truncated" when used in connection with TFmeans that the particular TF construct is lacking certain amino acidsequences. The term truncated thus means Tissue Factor constructs ofshorter length, and differentiates these compounds from other TissueFactor constructs that have reduced membrane association or binding.Although modified but substantially full-length TFs may thus beconsidered as functional equivalents of truncated TFs ("functionallytruncated"), the term "truncated" is used herein in its classical senseto mean that the TF molecule is rendered membrane-binding deficient byremoval of sufficient amino acid sequences to effect this change inproperty.

Accordingly, a truncated TF protein or polypeptide is one that differsfrom native TF in that a sufficient amount of the transmembrane aminoacid sequence has been removed from the molecule, as compared to thenative Tissue Factor. A "sufficient amount" in this context is an amountof transmembrane amino acid sequence originally sufficient to enter theTF molecule in the membrane, or otherwise mediate functional membranebinding of the TF protein. The removal of such a "sufficient amount oftransmembrane spanning sequence" therefore creates a truncated TissueFactor protein or polypeptide deficient in phospholipid membrane bindingcapacity, such that the protein is substantially a soluble protein thatdoes not significantly bind to phospholipid membranes, and thatsubstantially fails to convert Factor VII to Factor VIIa in a standardTF assay, and yet retains so-called catalytic activity includingactivating Factor X in the presence of Factor VIIa. U.S. Pat. No.5,504,067 is specifically incorporated herein by reference for thepurposes of further describing such truncated Tissue Factor proteins.

The preparation of particular truncated Tissue Factor constructs isdescribed herein below. Preferably, the Tissue Factors for use in thepresent invention will generally lack the transmembrane and cytosolicregions (amino acids 220-263 of SEQ ID NO:12) of the protein. However,there is no need for the truncated TF molecules to be limited tomolecules of the length of 219 amino acids. Therefore, constructs ofbetween about 210 and about 230 amino acids in length may be used. Inparticular, the constructs may be about 210, 211, 212, 213, 214, 215,216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, orabout 230 amino acids in length. Naturally, it will be understood thatthe intention is to substantially delete the transmembrane region ofabout 23 amino acids from the truncated molecule. Therefore, intruncated TF constructs that are longer than about 218-222 amino acidsin length, the significant sequence portions thereafter will generallybe comprised of about the 21 amino acids that form the cytosolic domainof the native TF molecule. In this regard, the truncated TF constructsmay be between about 231, 232, 233, 234, 235, 236, 237, 238, 239, 240,or about 241 amino acids in length.

In certain preferred embodiments, tTF may be designated as theextracellular domain of mature Tissue Factor protein. Therefore, inexemplary preferred embodiments, tTF may have the amino acid sequence ofSEQ ID NO:1, comprising residues 1-219 of the mature protein (SEQ IDNO:12). SEQ ID NO:1 may be encoded by, for example, SEQ ID NO:10. Ofcourse, SEQ ID NO:1 is only an exemplary tTF and any Tissue Factorprotein derived from the nucleic acid sequence SEQ ID NO:11, or relatedsequences, which possesses the desirable properties of high affinitybinding to Factor VII or to Factor VIIa and possesses a generallyreduced procoagulation cofactor activity will be useful as disclosedherein.

ii. Dimeric Tissue Factor Constructs

Previously it has been shown that it is possible for native TissueFactor on the surface of J82 bladder carcinoma cells to exist as a dimer(Fair et al., 1987). The binding of one Factor VII or Factor VIIamolecule to one Tissue Factor molecule may also facilitate the bindingof another Factor VII or Factor VIIa to another Tissue Factor (Fair etal, 1987; Bach et al., 1986). Furthermore, Tissue Factor showsstructural homology to members of the cytokine receptor family(Edgington et al., 1991) some of which dimerize to form active receptors(Davies and Wlodawer, 1995). As such it is contemplated that thetruncated Tissue Factor compositions of the present invention may beuseful as dimers.

Accordingly, any of the truncated, mutated or otherwisecoagulation-deficient Tissue Factor constructs disclosed herein, or anequivalent thereof, may be prepared in a dimeric form for use in thepresent invention. As will be known to those of ordinary skill in theart, such TF dimers may be prepared by employing the standard techniquesof molecular biology and recombinant expression, in which two codingregions are prepared in-frame and expressed from an expression vector.Equally, various chemical conjugation technologies may be employed inconnection with the preparation of TF dimers. The individual TF monomersmay be derivatized prior to conjugation. All such techniques would bereadily known to those of skill in the art.

If desired, the Tissue Factor dimers or multimers may be joined via abiologically-releasable bond, such as a selectively-cleavable linker oramino acid sequence. For example, peptide linkers that include acleavage site for an enzyme preferentially located or active within atumor environment are contemplated. Exemplary forms of such peptidelinkers are those that are cleaved by urokinase, plasmin, thrombin,Factor IXa, Factor Xa, or a metalloproteinase, such as collagenase,gelatinase or stromelysin.

In certain embodiments, the Tissue Factor dimers may further comprise ahindered hydrophobic membrane insertion moiety, to later encourage thefunctional association of the Tissue Factor with the phospholipidmembrane, but only under certain defined conditions. As described in thecontext of the truncated Tissue Factors, hydrophobicmembrane-association sequences are generally stretches of amino acidsthat promote association with the phospholipid environment due to theirhydrophobic nature. Equally, fatty acids may be used to provide thepotential membrane insertion moiety. Such membrane insertion sequencesmay be located either at the N-terminus or the C-terminus of the TFmolecule, or generally appended at any other point of the molecule solong as their attachment thereto does not hinder the functionalproperties of the TF construct. The intent of the hindered insertionmoiety is that it remains non-functional until the TF constructlocalizes within the tumor environment, and allows the hydrophobicappendage to become accessible and even further promote physicalassociation with the membrane. Again, it is contemplated thatbiologically-releasable bonds and selectively-cleavable sequences willbe particularly useful in this regard, with the bond or sequence onlybeing cleaved or otherwise modified upon localization within the tumorenvironment and exposure to particular enzymes or other bioactivemolecules.

By way of example only, the inventors have constructed dimeric tTFcorresponding to a dimer of C'-cys-tTF₂₁₉ (dimer of SEQ ID NO:3); adimer of C'-cys-tTF₂₂₀ (dimer of SEQ ID NO:6); a dimer of C'-cys-tTF₂₂₁(dimer of SEQ ID NO:7); and a dimer of H₆ -N'-cys-tTF₂₁₉ (dimer of SEQID NO:2). However, it will now be understood that each of the foregoingsequences are exemplary and by no means limiting of the dimericstructures that may be created and used in accordance with the presentinvention.

iii. Tri and Multimeric Tissue Factor Constructs

In other embodiments the tTF constructs of the present invention may bemultimeric or polymeric. In this context a "polymeric construct"contains 3 or more Tissue Factor constructs of the present invention. A"multimeric or polymeric TF construct" is a construct that comprises afirst TF molecule or derivative operatively attached to at least asecond and a third TF molecule or derivative, and preferably, whereinthe resultant multimeric or polymeric construct is still deficient incoagulating activity as compared to wild-type TF. In preferredembodiments, the multimeric and polymeric TF constructs for use in thisinvention are multimers or polymers of truncated TF molecules, which maybe optionally combined with other coagulation-deficient TF constructs orvariants. The multimers may comprise between about 3 and about 20 suchTF molecules, with between about 3 and about 15 or about 10 beingpreferred and between about 3 and about 10 being most preferred.Naturally, TF multimers of at least about 3, 4, 5, 6, 7, 8, 9 or 10 orso are included within the present invention. The individual TF unitswithin the multimers or polymers may also be linked byselectively-cleavable peptide linkers or other biological-releasablebonds as desired. Again, as with the TF dimers discussed above, theconstructs may be readily made using either recombinant manipulation andexpression or using standard synthetic chemistry.

iv. Factor VII Activation Mutants

Even further TF constructs useful in context of the present inventionare those mutants deficient in the ability to activate Factor VII. Thebasis for the utility of such mutants lies in the fact that they arealso "coagulation-deficient". Such "Factor VII activation mutants" aregenerally defined herein as TF mutants that bind functional FactorVII/VIIa, proteolytically activate Factor X, but are substantially freefrom the ability to proteolytically activate Factor VII. Accordingly,such constructs are TF mutants that lack Factor VII activation activity.

The ability of such Factor VII activation mutants to function inpromoting tumor-specific coagulation is based upon both the localizationof the TF construct to tumor vasculature, and the presence of FactorVIIa at low levels in plasma. Upon administration of such a Factor VIIactivation mutant, the mutant would generally localize within thevasculature of a vascularized tumor, as would any TF construct of theinvention. Prior to localization, the TF mutant would be generallyunable to promote coagulation in any other body sites, on the basis ofits inability to convert Factor VII to Factor VIIa. However, uponlocalization and accumulation within the tumor region, the mutant willthen encounter sufficient Factor VIIa from the plasma in order toinitiate the extrinsic coagulation pathway, leading to tumor-specificthrombosis.

As is developed more fully below, the most preferred use of the FactorVII activation mutants is in combination with the co-administration ofFactor VIIa. Although useful in and of themselves, as described above,such mutants will generally have less than optimal activity given thatFactor VIIa is known to be present in plasma only at low levels (about 1ng/ml, in contrast to about 500 ng/ml of Factor VII in plasma; U.S. Pat.Nos. 5,374,617; 5,504,064; and 5,504,067). Therefore, theco-administration of exogenous Factor VIIa along with the Factor VIIactivation mutant is considerably preferred over the administration ofthe mutants alone. In that these mutants are expected to have almost noside effects, their combined use with simultaneous, preceding orsubsequent administration of Factor VIIa is a particularly advantageousaspect of the present invention.

Any one or more of a variety of Factor VII activation mutants may beprepared and used in connection with either aspect of the presentinvention. There is a significant amount of scientific knowledgeconcerning the recognition sites on the TF molecule for Factor VII/VIIa.By way of example only, one may refer to the articles by Ruf andEdgington (1991a), Ruf et at. (1992c), and to WO 94/07515 and WO94/28017, each specifically incorporated herein by reference for furtherguidance on these matters. It will thus be understood that the FactorVII activation region generally lies between about amino acid 157 andabout amino acid 167 of the TF molecule. However, it is contemplatedthat residues outside this region may also prove to be relevant to theFactor VII activating activity, and one may therefore considerintroducing mutations into any one or more of the residues generallylocated between about amino acid 106 and about amino acid 209 of the TFsequence (WO 94/07515). In terms of the preferred region, one maygenerally consider mutating any one or more of amino acids 147, 152,154, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166 and/or 167.With reference to the generally preferred candidate mutations outsidethis region, one may refer to the following amino acid substitutions:S16, T17, S39, T30, S32, D34, V67, L104, B105, T106, R131, R136, V145,V146, F147, V198, N199, R200 and K201, with amino acids A34, E34 and R34also being considered (WO 94/28017).

As mentioned, preferably the Tissue Factors are rendered deficient inthe ability to activate Factor VII by altering one or more amino acidsfrom the region generally between about position 157 and about position167 in the amino acid sequence, when referring to SEQ ID NO:12.Exemplary mutants are those wherein Trp at position 158 is changed toArg (SEQ ID NO:8); wherein Ser at position 162 is changed to Ala;wherein Gly at position 164 is changed to Ala (SEQ ID NO:9); and thedouble mutant wherein Trp at position 158 is changed to Arg and Ser atposition 162 is changed to Ala. Of course these are exemplary mutationsand it is envisioned that any Tissue Factor mutant having an alteredamino acid composition that has the desirable characteristic of bindingto Factor VII/VIIa but not activating the coagulation cascade will beuseful in the context of the present invention.

A4. Quantitative In Vitro Assessment of Coagulant Deficiency

The Tissue Factor constructs of the present invention, whether they aretruncated, mutated, truncated and mutated, dimeric, multimeric,conjugated to inert carriers to increase their half-life, or anycombination of the foregoing, are each coagulation-deficient as comparedto native, wild-type Tissue Factor. By the term "coagulation-deficient",as used herein, is meant that the TF constructs have an impaired abilityto promote coagulation such that their administration into the systemiccirculation of an animal or human patient does not lead to significantside effects or limiting toxicity. A TF construct can be readilyanalyzed in order to determine whether it meets this definition, simplyby conducting a test in an experimental animal. However, the followingdetailed guidance is provided to assist those of skill in the art in theprior characterization and selection of appropriate candidatescoagulation-deficient TF constructs, in order that any experimentalanimal studies may be conducted efficiently and cost-efficiently.

In quantitative terms, the coagulation-deficient TFs will be 100-fold ormore less active than full length, native TF, that is, they will be100-fold or more less able to induce coagulation of plasma than is fulllength, native TF when tested in an appropriate phospholipidenvironment.

More preferably, the impaired TFs should be 1,000-fold or more less ableto induce coagulation of plasma than is full length, wild type TF in anappropriate phospholipid environment; even more preferably, the TFsshould be 10,000-fold or more less able to induce coagulation of plasmathan full length, wild type TF in such an environment; and mostpreferably, the impaired TFs should be 100,000-fold or more less able toinduce coagulation of plasma than is full length, native TF in anappropriate phospholipid environment. It will be appreciated that this"100,000-fold" generally corresponds to one of the currently preferredconstructs, the truncated Tissue Factor of 219 amino acids in length(SEQ ID NO:1).

Inherent within the definition of "X-fold or more less able to inducecoagulation of plasma" is the concept that the subject TF undergoinginvestigation is still able to induce coagulation of plasma. Evidently,a TF that has been modified to render its completely unable to inducecoagulation will generally not be useful in the context of the presentinvention. TFs that are less active than wild-type TF in the controlled,phospholipid assays by about 500,000-fold are still contemplated to haveutility in connection herewith. Similarly, all TF variants and mutantsthat are between about 500,000-fold and about 1,000,000-fold less ableto induce coagulation of plasma than is full length, native TF in anappropriate phospholipid environment are still envisioned to haveutility in certain embodiments. However, it is generally considered that1,000,000-fold (10⁶) impairment of activity will generally be about theleast active that one would consider for use in the present invention.Furthermore, those TF constructs that are towards the less active end ofthe stated range may find most utility in connection with certain,defined treatments regimens, or in combined therapies. The choice ofparticular TF variant and therapeutic strategy will be readilydetermined by one of ordinary skill in the art.

Notwithstanding that there will be certain preferred and/or optimal usesand combinations of the various TF elements, the coagulation-deficientTFs for use in the present invention will generally be between about100-fold and about 1,000,000-fold less active than wild-type TF; morepreferably, will be between about 1,000-fold and about 100,000-fold lessactive; and may be categorized as less active by any number within thestated ranges, including by about 10,000-fold. The ranges themselves mayalso be varied between about 1,000-fold and 1,000,000-fold, or betweenabout 10,000-fold and 500,000-fold, or such like.

Any one or more of a number of in vitro plasma coagulation activityassays may be employed in connection with the quantitative testing ofcandidate coagulation-deficient Tissue Factors. For example, one methodof conducting an innate plasma coagulation activity assay is as follows:

1) add about 50 μl plasma (human or mouse) to plastic tubes at about 37°C.;

2) add about 50 μl of relipidated full length TF (preferably from acommercial source, such as American Diagnostics Inc., Greenwich, Conn.)at a range of concentrations in a suitable buffer such as calcium-freephosphate or HEPES buffered saline, pH 7.4 at 37° C. To other tubes addthe Tissue Factor candidate truncated or mutant version at a range ofconcentrations in the same buffer.

3) add about 50 μl 30 mM CaCl₂ at about 37° C.;

4) record the time for the first fibrin strands to form; and

5) Construct a standard curve of full length TF concentration (mol perliter) against coagulation time. Construct a curve of the candidatemutant TF concentration (mol per liter) against coagulation time.Calculate the difference in activity between the full length TF and the"test" TF by comparing the concentration of each needed to give acoagulation time equivalent to about half the maximal decrease incoagulation time. The "test" mutant TF should be more than 100-fold lessable than the full length TF on a molar basis to induce coagulation ofplasma.

Variations of this type of assay can be conducted, as would be evidentto one of ordinary skill in the art. For example, one may conduct theassays based upon the attachment of Tissue Factor and the candidateTissue Factor construct to a cell membrane or phospholipid surface, forexample, using an antibody or other ligand to effect such an attachment.In such assays, the candidate or test truncated TF or TF mutant shouldbe greater than 100-fold less effective at inducing coagulation ofplasma than wild-type TF, when it is attached by means of an antibody orother ligand to a cell membrane or phospholipid surface. With TissueFactor mutants that do not allow Factor VII to be efficiently convertedto Factor VIIa, it may be necessary to add Factor VIIa to the plasma toobtain this level of activity. In an exemplary assay, such activity canbe measured using the following method:

1) Cells such as A20 mouse lymphoma cells (I-A^(d) positive) (e.g.,4×10⁶ cells/ml, 50 μl) in a buffer such as phosphate-buffered saline areincubated for about 1 hour at about room temperature with anattachment-promoting agent, such as a bispecific antibody (50 μg/ml, 25μl), e.g., in terms of A20 cells, consisting of a Fab' arm of anantibody such as the B21-2 antibody directed against I-A^(d), linked toFab' arm of an antibody such as the 10H10 antibody directed against anon-inhibitory epitope on TF;

2) Prepare an identical set of tubes which contain cells, but nobispecific antibody or other tethering agent;

3) Wash the cells effectively, e.g., twice at room temperature, andresuspend the cells in about 50 μl of phosphate buffered saline.

4) Add varying concentrations of the candidate TF mutants in phosphatebuffered saline (about 50 μl) at about room temperature. The bispecificantibody or other tethering agent captures the TF mutant and brings itinto close approximation to the cell surface. Factor VIIa (1-10 nM) isadded in addition to the TF mutant when it is desired to determine theactivity in the presence of Factor VIIa. The total volume per tube isadjusted to about 150 μl with phosphate buffered saline. Tubes areincubated for about 1 h at about room temperature;

5) Warm the cells to about 37° C.

6) Add calcium chloride (about 50 mM, 50 μl) and citrated mouse or humanplasma (about 50 μl) at about 37° C.

7) Record the time for the first fibrin strands to form; and

8) Plot coagulation time (in seconds) against concentration of TF mutant(mol per liter) for cells coated or not coated with tethering agent,e.g., bispecific antibody. The TF mutant concentration that gives acoagulation time equivalent to approximately half the maximal decreasein coagulation time (usually 50-100 sec) is calculated. The enhancementin coagulation activity given by the bispecific antibody is calculatedand should be in excess of 100-fold.

It is envisioned that candidate TF compositions prepared by the presentinvention may be tested using assays similar to those described above toconfirm that their functionality has been maintained, but that theirability to promote coagulation has been impaired by at least therequired amount of about 100-fold and preferably by about 1,000-fold,more preferably by about 10,000-fold, and most preferably by about100,000-fold.

In embodiments where it is contemplated that an additional agent shouldultimately be used in combination with the candidatecoagulation-deficient TF, it is important that the additional factor oragent be included in the in vitro assay. A particularly relevant exampleis the analysis of a Factor VII activation mutant, which shouldpreferably be analyzed in conjunction with the addition of Factor VIIa.However, Factor VIIa is not the only additional component that may betested in this manner. In general, the additional agents may be termed"additional candidates". To identify an additional candidate, or tooptimize preferred amounts of the candidates for use in the presentinvention, one would conduct assays such as those described above inparallel. That is, one would measure or determine the coagulation in theabsence of the additional candidate, and then one would add thecandidate substance to the composition and re-determine the time and/orextent of the blood coagulation. An additional candidate substance thatfunctions in combination with a TF mutant or variant to result in anoverall level of coagulation that is between about 100-fold and about1,000,000-fold less than that observed with native TF will again be anappropriate combination for use in the context of the present invention.

Those of ordinary skill in the art will understand that each of theforegoing in vitro assays and variations thereof are relatively simpleto establish and perform. In this manner, a panel of candidates TFvariants and combinations of TFs with other agents can be tested and themost promising candidates selected for further studies, particularly forexperimental testing in an animal or human trial.

Notwithstanding that the foregoing assays are believed to beparticularly useful in connection with the present invention, the invitro testing contemplated for use herewith is not limited to suchassays. Accordingly, one may conduct any type of coagulation orprocoagulation assay that one desires. For example, for further detailsregarding tTF and procoagulation assays, the skilled practitioner isreferred to U.S. Pat. Nos. 5,437,864; 5,223,427; and 5,110,730 and PCTpublication numbers WO 94/28017; WO 94/05328; and WO 94/07515, each ofwhich are specifically incorporated by reference herein for the purposesof even further supplementing the present disclosure in regard toassays.

A5. Confirmatory in Vivo Studies

It will be understood by those of skill in the art that the candidatecoagulation-deficient Tissue Factor mutants, variants or combinations ofsuch with additional agents, should generally be tested in an in vivosetting prior to use in a human subject. Such pre-clinical testing inanimals is routine in the art. To conduct such confirmatory tests, allthat is required is an art-accepted animal model of the disease inquestion, such as an animal bearing a solid tumor. Any animal may beused in such a context, such as, e.g., a mouse, rat, guinea pig,hamster, rabbit, dog, chimpanzee, or such like. In the context of cancertreatment, studies using small animals such as mice are widely acceptedas being predictive of clinical efficacy in humans, and such animalmodels are therefore preferred in the context of the present inventionas they are readily available and relatively inexpensive, at least incomparison to other experimental animals.

The manner of conducting an experimental animal test will bestraightforward to those of ordinary skill in the art. All that isrequired to conduct such a test is to establish equivalent treatmentgroups, and to administer the test compounds to one group while variouscontrol studies are conducted in parallel on the equivalent animals inthe remaining group or groups. One monitors the animals during thecourse of the study and, ultimately, one sacrifices the animals toanalyze the effects of the treatment.

One of the most useful features of the present invention is itsapplication to the treatment of vascularized tumors. Accordingly,anti-tumor studies can be conducted to determine the specific thrombosiswithin the tumor vasculature and the anti-tumor effects overall. As partof such studies, the specificity of the effects should also bemonitored, including evidence of coagulation in other vessels andtissues and the general well being of the animals should be carefullymonitored.

In the context of the treatment of solid tumors, it is contemplated thateffective TF constructs and effective amounts of the constructs will bethose constructs and amounts that generally result in at least about 10%of the vessels within a vascularized tumor exhibiting thrombosis, in theabsence of significant thrombosis in non-tumor vessels; preferably,thrombosis will be observed in at least about 20%, about 30%, about 40%,or about 50% also of the blood vessels within the solid tumor mass,without significant non-localized thrombosis. In the treatment of largetumors, such positive effects have been routinely observed by thepresent inventors. Indeed, tumors have been analyzed in which at leastabout 60%, about 70%, about 80%, about 85%, about 90%, about 95% or evenup to and including about 99% of the tumor vessels have becomethrombotic. Naturally, the more vessels that exhibit thrombosis, themore preferred is the treatment, so long as the effect remains specific,relatively specific or preferential to the tumor-associated vasculatureand so long as coagulation is not apparent in other tissues to a degreesufficient to cause significant harm to the animal.

Following the induction of thrombosis within the tumor blood vessels,the surrounding tumor tissues become necrotic. The successful use of theconstructs of the invention, or the doses thereof, can thus also beassessed in terms of the expanse of the necrosis induced specifically inthe tumor. Again, the expanse of cell death in the tumor will beassessed relative to the maintenance of healthy tissues in all otherareas of the body. TF agents, combinations or optimal doses will havetherapeutic utility in accordance with the present invention when theiradministration results in at least about 10% of the tumor tissuebecoming necrotic (10% necrosis). Again, it is preferable to elicit atleast about 20%, about 30%, about 40% or about 50% necrosis in the tumorregion, without significant, adverse side-effects. Such beneficialeffects have again been observed by the present inventors. Naturally, itwill be preferable to use constructs and doses capable of inducing atleast about 60%, about 70%, about 80%, about 85%, about 90%, about 95%up to and including 99% tumor necrosis, so long as the constructs anddoses used do not result in significant side effects or other untowardreactions in the animal.

All of the above determinations can be readily made and properlyassessed by those of ordinary skill in the art. For example, attendantscientists and physicians can utilize such data from experimentalanimals in the optimization of appropriate doses for human treatment. Insubjects with advanced disease, a certain degree of side effects can betolerated. However, patients in the early stages of disease can betreated with more moderate doses in order to obtain a significanttherapeutic effect in the absence of side effects. The effects observedin such experimental animal studies should preferably be statisticallysignificant over the control levels and should be reproducible fromstudy to study.

Those of ordinary skill in the art will further understand that TFconstructs, combinations and doses that result in tumor-specificthrombosis and necrosis towards the lower end of the effective rangesquoted above may nonetheless still be useful in connection with thepresent invention. For example, in embodiments where a continuedapplication of the active agents is contemplated, an initial dose of aconstruct that results in only about 10% thrombosis and/or necrosis willnonetheless be useful, particularly as it is often observed that thisinitial reduction "primes" the tumor to further destructive assault uponsubsequent re-application of the therapy. In any event, even if upwardsof about 40% or so tumor inhibition is not ultimately achieved (which isthe general goal), it will be understood that any induction ofthrombosis and necrosis is nonetheless useful in that it represents anadvance over the state of the patients prior to treatment.

As discussed above in connection with the in vitro test system, it willnaturally be understood that combinations of agents intended for usetogether should be tested and optimized together. By way of exampleonly, the Factor VIIa activation mutant of the present invention fallinto this category and should generally be tested in conjunction withthe simultaneous, prior or subsequent administration of exogenous FactorVIIa. Similarly, the individual TF constructs of the present inventioncan be straightforwardly analyzed in combination with one or morechemotherapeutic drugs, immunotoxins, coaguligands or such like.Analysis of the combined effects of such agents would be determined andassessed according to the guidelines set forth above.

A6. Biologically Functional Equivalents

As discussed, tTF compositions useful in the present invention are thosethat will generally promote coagulation at least 100-fold lesseffectively than wild type TF. In other embodiments the tTF promotescoagulation at least 10³ fold less effectively, in yet other embodimentsthe tTF promotes coagulation at least 10⁴ or even 10⁵ times lesseffectively than wild type TF, with TFs that are about 10⁶ times or soless active than wild type TF being about the intended minimum activityrequired.

Exemplary TFs are those that lack the transmembrane and cytosolic region(amino acids 220-263). An exemplary tTF of the present invention isgiven in SEQ ID NO:1 and contains amino acids 1-219 of wild type TissueFactor (SEQ ID NO:12). Of course this is only an exemplary tTF and othertTF construct are contemplated, for example, a construct comprisingamino acids 1-220; 2-219, 3-219 or any other truncation of SEQ ID NO:12that renders the molecule lacking in the transmembrane domain and/orcytosolic domains of wild type Tissue Factor otherwise results in afunctionally comparative molecule. Mutants are also contemplated, asdescribed in detail above.

Using the detailed guidance provided above, even further equivalents ofthe TFs can be made. Modifications and changes may be made in thestructure of TF and still obtain a molecule having like or otherwisedesirable characteristics. For example certain amino acids maysubstituted for other amino acids in a protein structure withoutappreciable loss of interactive binding capacity, such as, for example,binding to Factor VIIa. Since it is the interactive capacity and natureof a protein that defines that protein's biological functional activity,certain amino acid sequence substitutions can be made in a proteinsequence (or of course, the underlying DNA sequence) and nevertheless,obtain a protein with like (agonistic) properties. It is thuscontemplated that various changes may be made in the sequence of TF (SEQID NO:12) proteins and peptides (or underlying DNA sequence, SEQ IDNO:11) without appreciable loss of their biological utility or activity.

It also is well understood by the skilled artisan that, inherent in thedefinition of a biologically functional equivalent protein or peptide,is the concept that there is a limit to the number of changes that maybe made within a defined portion of the molecule and still result in amolecule with an acceptable level of equivalent biological activity.Biologically functional equivalent peptides are thus defined herein asthose peptides in which certain, not most or all, of the amino acids maybe substituted. Of course, a plurality of distinct proteins/peptideswith different substitutions may easily be made and used in accordancewith the invention.

Amino acid substitutions are generally based on the relative similarityof the amino acid side-chain substituents, for example, theirhydrophobicity, hydrophilicity, charge, size, and the like. An analysisof the size, shape and type of the amino acid side-chain substituentsreveals that arginine, lysine and histidine are all positively chargedresidues; that alanine, glycine and serine are all a similar size; andthat phenylalanine, tryptophan and tyrosine all have a generally similarshape. Therefore, based upon these considerations, arginine, lysine andhistidine; alanine, glycine and serine; and phenylalanine, tryptophanand tyrosine; are defined herein as biologically functional equivalents.

In making more quantitative changes, the hydropathic index of aminoacids may be considered. Each amino acid has been assigned a hydropathicindex on the basis of their hydrophobicity and charge characteristics,these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8);phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9);alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8);tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2);glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5);lysine (-3.9); and arginine (-4.5).

The importance of the hydropathic amino acid index in conferringinteractive biological function on a protein is generally understood inthe art (Kyte and Doolittle, 1982, incorporated herein by reference). Itis known that certain amino acids may be substituted for other aminoacids having a similar hydropathic index or score and still retain asimilar biological activity. In making changes based upon thehydropathic index, the substitution of amino acids whose hydropathicindices are within ±2 is preferred, those which are within ±1 areparticularly preferred, and those within ±0.5 are even more particularlypreferred.

It is thus understood that an amino acid can be substituted for anotherhaving a similar hydrophilicity value and still obtain a biologicallyequivalent protein. As detailed in U.S. Pat. No. 4,554,101 (incorporatedherein by reference), the following hydrophilicity values have beenassigned to amino acid residues: arginine (+3.0); lysine (+3.0);aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3); asparagine(+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline(-0.5±1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine(-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine(-2.3); phenylalanine (-2.5); tryptophan (-3.4).

In making changes based upon hydrophilicity values, the substitution ofamino acids whose hydrophilicity values are within ±2 is preferred,those which are within ±1 are particularly preferred, and those within±0.5 are even more particularly preferred.

B. Tissue Factor Polynucleotides

B1. DNA Segments

The polynucleotides encoding the TFs of the present invention may encodean entire TF protein, so long as it is coagulation-deficient, afunctional TF protein domain, or any TF polypeptide, mutant or variantin accordance with the detailed guidance set forth herein. Whether onedesires to prepare a truncated TF, a mutant TF or a truncated andmutated TF, the underlying useful DNA segment and gene will be generallythe same. In that the human DNA for the entire TF molecule is available,it will generally be preferred to use this human construct given thatclinical treatment in humans is intended. However, the use of other TFgenes is by no means excluded, so long as the protein produced does notelicit significant immunological or other untoward reactions uponadministration to a human patient. The methods and compositionsdescribed in U.S. Pat. No. 5,110,730 are specifically incorporatedherein by reference for the purposes of even further supplementingApplicants' disclosure concerning the genes and DNA segments for useherewith.

The polynucleotides may be derived from genomic DNA, i.e., cloneddirectly from the genome of a particular organism. In other embodiments,however, the polynucleotides may be complementary DNA (cDNA). cDNA isDNA prepared using messenger RNA (mRNA) as template. Thus, a cDNA doesnot contain any interrupted coding sequences and usually contains almostexclusively the coding region(s) for the corresponding protein. In otherembodiments, the polynucleotide may be produced synthetically. As isknown to those of skill in the art, it is generally preferred to use acDNA construct in the recombinant expression given that such constructsare easier to manipulate and use. The use of longer, genomic clones upto and including full length sequences are, however, by no meansexcluded.

Although a surprising feature of the present invention is that the TFconstructs preferentially are specifically localized in the vasculatureof a solid tumor and induce specific anti-tumor effects therein, it isalso contemplated that the TF proteins and polypeptides may be deliveredto the tumor environment using a recombinant vector that expresses theTF products. Such "gene therapy" approaches to cancer treatment can bereadily practiced by reference to certain scientific referencesconcerning appropriate constructs and protocols. By way of example only,one may use a viral vector, such as a retroviral vector, herpes simplexvirus, HSV (U.S. Pat. No. 5,288,641), cytomegalovirus; adeno-associatedvirus, AAV (U.S. Pat. No. 5,139,941); and/or an adenoviral vector.

The genomic human DNA sequence for TF is provided in SEQ ID NO:11, withthe corresponding amino acid sequence being provided in SEQ ID NO:12.Should one desire to express Factor VII, the DNA and amino acidsequences are provided in SEQ ID NO:13 and SEQ ID NO:14, respectively.

It is contemplated that natural variants of TF exist that have differentsequences than those disclosed herein. Thus, the present invention isnot limited to use of the provided polynucleotide sequence for TF but,rather, includes use of any naturally-occurring variants. The presentinvention also encompasses chemically synthesized mutants of thesesequences, intelligently designed following an application of thestructural and quantitated functional considerations detailed above.

Another kind of sequence variant results from codon variation. Becausethere are several codons for most of the 20 normal amino acids, manydifferent DNA's can encode the TF. Reference to Table I will allow suchvariants to be identified.

                  TABLE I                                                         ______________________________________                                        Amino Acids    Codons                                                         ______________________________________                                        Alanine  Ala    A      GCA  GCC  GCG  GCU                                     Cysteine Cys    C      UGC  UGU                                               Aspartic acid                                                                          Asp    D      GAC  GAU                                               Glutamic acid                                                                          Glu    E      GAA  GAG                                               Phenylalanine                                                                          Phe    F      UUC  UUU                                               Glycine  Gly    G      GGA  GGC  GGG  GGU                                     Histidine                                                                              His    H      CAC  CAU                                               Isoleucine                                                                             Ile    I      AUA  AUC  AUU                                          Lysine   Lys    K      AAA  AAG                                               Leucine  Leu    L      UUA  UUG  CUA  CUC  CUG  CUU                           Methionine                                                                             Met    M      AUG                                                    Asparagine                                                                             Asn    N      AAC  AAU                                               Proline  Pro    P      CCA  CCC  CCG  CCU                                     Glutamine                                                                              Gln    Q      CAA  CAG                                               Arginine Arg    R      AGA  AGG  CGA  CGC  CGG  CGU                           Serine   Ser    S      AGC  AGU  UCA  UCC  UCG  UCU                           Threonine                                                                              Thr    T      ACA  ACC  ACG  ACU                                     Valine   Val    V      GUA  GUC  GUG  GUU                                     Tryptophan                                                                             Trp    W      UGG                                                    Tyrosine Tyr    Y      UAC UAU                                                ______________________________________                                    

B2. Mutagenesis

Site-specific mutagenesis is a technique useful in the preparation ofindividual peptides, or biologically functional equivalent proteins orpeptides, through specific mutagenesis of the underlying DNA. Thetechnique further provides a ready ability to prepare and test sequencevariants, incorporating one or more of the foregoing considerations, byintroducing one or more nucleotide sequence changes into the DNA.Site-specific mutagenesis allows the production of mutants through theuse of specific oligonucleotide sequences which encode the DNA sequenceof the desired mutation, as well as a sufficient number of adjacentnucleotides, to provide a primer sequence of sufficient size andsequence complexity to form a stable duplex on both sides of thedeletion junction being traversed. Typically, a primer of about 17 to 25nucleotides in length is preferred, with about 5 to 10 residues on bothsides of the junction of the sequence being altered.

The technique of site-specific mutagenesis is well known in the art. Aswill be appreciated, the technique typically employs a bacteriophagevector that exists in both a single stranded and double stranded form.Typical vectors useful in site-directed mutagenesis include vectors suchas the M13 phage. These phage vectors are commercially available andtheir use is generally well known to those skilled in the art. Doublestranded plasmids are also routinely employed in site directedmutagenesis, which eliminates the step of transferring the gene ofinterest from a phage to a plasmid.

In general, site-directed mutagenesis is performed by first obtaining asingle-stranded vector, or melting of two strands of a double strandedvector which includes within its sequence a DNA sequence encoding thedesired protein. An oligonucleotide primer bearing the desired mutatedsequence is synthetically prepared. This primer is then annealed withthe single-stranded DNA preparation, and subjected to DNA polymerizingenzymes such as E. coli polymerase I Klenow fragment, in order tocomplete the synthesis of the mutation-bearing strand. Thus, aheteroduplex is formed wherein one strand encodes the originalnon-mutated sequence and the second strand bears the desired mutation.This heteroduplex vector is then used to transform appropriate cells,such as E. coli cells, and clones are selected that include recombinantvectors bearing the mutated sequence arrangement.

The preparation of sequence variants of the selected gene usingsite-directed mutagenesis is provided as a means of producingpotentially useful species and is not meant to be limiting, as there areother ways in which sequence variants of genes may be obtained. Forexample, recombinant vectors encoding the desired gene may be treatedwith mutagenic agents, such as hydroxylamine, to obtain sequencevariants. Suitable techniques are also described in U.S. Pat. No.4,888,286, incorporated herein by reference.

Although the foregoing methods are suitable for use in mutagenesis, theuse of the polymerase chain reaction (PCR™) is generally now preferred.This technology offers a quick and efficient method for introducingdesired mutations into a given DNA sequence. The following textparticularly describes the use of PCR™ to introduce point mutations intoa sequence, as may be used to change the amino acid encoded by the givensequence. Adaptations of this method are also suitable for introducingrestriction enzyme sites into a DNA molecule.

In this method, synthetic oligonucleotides are designed to incorporate apoint mutation at one end of an amplified segment. Following PCR™, theamplified fragments are blunt-ended by treating with Klenow fragments,and the blunt-ended fragments are then ligated and subcloned into avector to facilitate sequence analysis.

To prepare the template DNA that one desires to mutagenize, the DNA issubcloned into a high copy number vector, such as pUC19, usingrestriction sites flanking the area to be mutated. Template DNA is thenprepared using a plasmid miniprep. Appropriate oligonucleotide primersthat are based upon the parent sequence, but which contain the desiredpoint mutation and which are flanked at the 5' end by a restrictionenzyme site, are synthesized using an automated synthesizer. It isgenerally required that the primer be homologous to the template DNA forabout 15 bases or so. Primers may be purified by denaturingpolyacrylamide gel electrophoresis, although this is not absolutelynecessary for use in PCR™. The 5' end of the oligonucleotides shouldthen be phosphorylated.

The template DNA should be amplified by PCR™, using the oligonucleotideprimers that contain the desired point mutations. The concentration ofMgCl₂ in the amplification buffer will generally be about 15 mM.Generally about 20-25 cycles of PCR™ should be carried out as follows:denaturation, 35 sec. at 95° C.; hybridization, 2 min. at 50° C.; andextension, 2 min. at 72° C. The PCR™ will generally include a last cycleextension of about 10 min. at 72° C. After the final extension step,about 5 units of Klenow fragments should be added to the reactionmixture and incubated for a further 15 min. at about 30° C. Theexonuclease activity of the Klenow fragments is required to make theends flush and suitable for blunt-end cloning.

The resultant reaction mixture should generally be analyzed bynondenaturing agarose or acrylamide gel electrophoresis to verify thatthe amplification has yielded the predicted product. One would thenprocess the reaction mixture by removing most of the mineral oils,extracting with chloroform to remove the remaining oil, extracting withbuffered phenol and then concentrating by precipitation with 100%ethanol. Next, one should digest about half of the amplified fragmentswith a restriction enzyme that cuts at the flanking sequences used inthe oligonucleotides. The digested fragments are purified on a lowgelling/melting agarose gel.

To subcdone the fragments and to check the point mutation, one wouldsubclone the two amplified fragments into an appropriately digestedvector by blunt-end ligation. This would be used to transform E. coli,from which plasmid DNA could subsequently be prepared using a miniprep.The amplified portion of the plasmid DNA would then be analyzed by DNAsequencing to confirm that the correct point mutation was generated.This is important as Taq DNA polymerase can introduce additionalmutations into DNA fragments.

The introduction of a point mutation can also be effected usingsequential PCR™ steps. In this procedure, the two fragments encompassingthe mutation are annealed with each other and extended by mutuallyprimed synthesis. This fragment is then amplified by a second PCR™ step,thereby avoiding the blunt-end ligation required in the above protocol.In this method, the preparation of the template DNA, the generation ofthe oligonucleotide primers and the first PCR™ amplification areperformed as described above. In this process, however, the chosenoligonucleotides should be homologous to the template DNA for a stretchof between about 15 and about 20 bases and must also overlap with eachother by about 10 bases or more.

In the second PCR™ amplification, one would use each amplified fragmentand each flanking sequence primer and carry PCR™ for between about 20and about 25 cycles, using the conditions as described above. One wouldagain subclone the fragments and check that the point mutation wascorrect by using the steps outlined above.

In using either of the foregoing methods, it is generally preferred tointroduce the mutation by amplifying as small a fragment as possible. Ofcourse, parameters such as the melting temperature of theoligonucleotide, as will generally be influenced by the GC content andthe length of the oligo, should also be carefully considered. Theexecution of these methods, and their optimization if necessary, will beknown to those of skill in the art, and are further described in variouspublications, such as Current Protocols in Molecular Biology, 1995,incorporated herein by reference.

B3. Expression Constructs and Protein Production

Throughout this application, the term "expression construct" is meant toinclude any type of genetic construct containing a nucleic acid codingfor a gene product in which part or all of the nucleic acid encodingsequence is capable of being transcribed. The transcript will generallybe translated into a protein. Thus, expression preferably includes bothtranscription of a TF gene and translation of a TF mRNA into a TFprotein product.

A technique often employed by those skilled in the art of proteinproduction today is to obtain a so-called "recombinant" version of theprotein, to express it in a recombinant cell and to obtain the proteinfrom such cells. These techniques are based upon the "cloning" of a DNAmolecule encoding the protein from a DNA library, i.e., on obtaining aspecific DNA molecule distinct from other portions of DNA. This can beachieved by, for example, cloning a cDNA molecule, or cloning agenomic-like DNA molecule. Techniques such as these would be appropriatefor the production of particular TF compositions in accordance with thepresent invention. Recombinant fusion proteins are discussed in furtherdetail herein below, and in U.S. Pat. No. 5,298,599, incorporated hereinby reference for the purposes of further exemplification of fusionprotein production and use.

For the expression of TF, once a suitable (full-length if desired) cloneor clones have been obtained, whether they be cDNA based or genomic, onemay proceed to prepare an expression system for the recombinantpreparation of TFs. The engineering of DNA segment(s) for expression ina prokaryotic or eukaryotic system may be performed by techniquesgenerally known to those of skill in recombinant expression. It isbelieved that virtually any expression system may be employed in theexpression of these proteins.

Such proteins may be successfully expressed in eukaryotic expressionsystems, e.g., CHO cells, as described by Rehemtulla et al. (1991),however, it is envisioned that bacterial expression systems, such as E.coli pQE-60 will be particularly useful for the large-scale preparationand subsequent purification of the proteins or peptides. cDNAs for TFmay be expressed in bacterial systems, with the encoded proteins beingexpressed as fusions with β-galactosidase, ubiquitin, Schistosomajaponicum glutathione S-transferase, and the like. It is believed thatbacterial expression will have advantages over eukaryotic expression interms of ease of use and quantity of materials obtained thereby. Thetechniques of U.S. Pat. Nos. 5,298,599 and 5,346,991 are alsoincorporated herein by reference to even further supplement the solubleTissue Factor production methods disclosed herein, with U.S. Pat. No.5,346,991 being particularly incorporated for the purposes of evenfurther supplementing the disclosure regarding the creation andproduction of Tissue Factor mutants and variants.

In order for the construct to effect expression of a TF transcript, thepolynucleotide encoding the TF polynucleotide will be under thetranscriptional control of a promoter. A "promoter" refers to a DNAsequence recognized by the synthetic machinery of the host cell, orintroduced synthetic machinery, that is required to initiate thespecific transcription of a gene. The phrase "under transcriptionalcontrol" means that the promoter is in the correct location in relationto the polynucleotide to control RNA polymerase initiation andexpression of the polynucleotide. The term promoter will be used here torefer to a group of transcriptional control modules that are clusteredaround the initiation site for RNA polymerase II.

In terms of microbial expression, U.S. Pat. Nos. 5,583,013; 5,221,619;4,785,420; 4,704,362; and 4,366,246 are incorporated herein by referencefor the purposes of even further supplementing the present disclosure inconnection with the expression of genes in recombinant host cells.

B4. Purification of Tissue Factor and Related Compositions

Once the peptides have been expressed they may be isolated and purifiedusing protein purification techniques well known to those of skill inthe art. Such compositions will be employed alone or in combination withantibodies, chemotherapeutics and effector ligands as therapeutic agentsin the treatment of tumors as detailed herein below. Exemplary peptidesof the present invention are shown in SEQ ID NO: 1-SEQ ID NO:9, ofcourse it is understood that these are only exemplary and any mutations,alterations or naturally occurring variants of these sequences are alsocontemplated to be useful in conjunction with the present invention.

Protein purification techniques are well known to those of skill in theart. These techniques tend to involve the fractionation of the cellularmilieu to separate the protein of interest from other components of themixture. Analytical methods particularly suited to the preparation of apure peptide are ion-exchange chromatography, exclusion chromatography,polyacrylamide gel electrophoresis, isoelectric focusing and the like. Aparticularly efficient method of purifying peptides is fast proteinliquid chromatography or even HPLC.

Various other techniques suitable for use in protein purification willbe well known to those of skill in the art. These include, for example,precipitation with ammonium sulfate, PEG, antibodies and the like or byheat denaturation, followed by centrifugation; chromatography steps suchas ion exchange, gel filtration, reverse phase, hydroxylapatite andaffinity chromatography; isoelectric focusing; gel electrophoresis; andcombinations of such and other techniques. As is generally known in theart, it is believed that the order of conducting the variouspurification steps may be changed, or that certain steps may be omitted,and still result in a suitable method for the preparation of asubstantially purified protein or peptide.

As disclosed herein in detail, the generally preferred techniques forpurifying expressed TF constructs for use in the present inventioninvolve the generation of a TF molecule that includes an affinitypurification tag and the use of an affinity separation matrix forobtaining the TF construct free from most or all contaminating species.Many such fusion protein tags are known to those of ordinary skill inthe art and such expression and separating protocols can be easilyexecuted. Technology is also available for cleaving the originalaffinity tag prior to use of the released protein or polypeptide, whichmay be effected by inserting a protease-sensitive linker between theaffinity tag and the protein of interest. Such methodology is indeedemployed in connection with aspects of the present invention. U.S. Pat.No. 5,298,599 is also instructive in this regard. However, it is alsoknown that many such tags do not impair the ability of the expressedprotein to carry out their biological functions, and removal of a tag isnot necessarily required prior to use of the TF construct in the presentinvention.

C. Pharmaceutical Compositions and Kits

Pharmaceutical compositions of the present invention will generallycomprise an effective amount of the tTF dissolved or dispersed in apharmaceutically acceptable carrier or aqueous medium.

The phrases "pharmaceutically or pharmacologically acceptable" refer tomolecular entities and compositions that do not produce an adverse,allergic or other untoward reaction when administered to an animal, or ahuman, as appropriate. As used herein, "pharmaceutically acceptablecarrier" includes any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents and the like. The use of such media and agents for pharmaceuticalactive substances is well known in the art. Except insofar as anyconventional media or agent is incompatible with the active ingredient,its use in the therapeutic compositions is contemplated. Supplementaryactive ingredients can also be incorporated into the compositions.

C1. Parenteral Formulations

The tTF of the present invention will often be formulated for parenteraladministration, e.g., formulated for injection via the intravenous,intramuscular, sub-cutaneous or other such routes, including directinstillation into a tumor or disease site. The preparation of an aqueouscomposition that contains a tumor-targeted coagulant agent as an activeingredient will be known to those of skill in the art in light of thepresent disclosure. Typically, such compositions can be prepared asinjectables, either as liquid solutions or suspensions; solid formssuitable for using to prepare solutions or suspensions upon the additionof a liquid prior to injection can also be prepared; and thepreparations can also be emulsified.

Solutions of the active compounds as free base or pharmacologicallyacceptable salts can be prepared in water suitably mixed with asurfactant, such as hydroxypropylcellulose. Dispersions can also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions; formulations including sesame oil,peanut oil or aqueous propylene glycol; and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi.

The tTF compositions can be formulated into a composition in a neutralor salt form. Pharmaceutically acceptable salts, include the acidaddition salts (formed with the free amino groups of the protein) andwhich are formed with inorganic acids such as, for example, hydrochloricor phosphoric acids, or such organic acids as acetic, oxalic, tartaric,mandelic, and the like. Salts formed with the free carboxyl groups canalso be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isopropylamine, trimethylamine, histidine, procaine and thelike.

The carrier can also be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetable oils. The proper fluidity can be maintained, forexample, by the use of a coating, such as lecithin, by the maintenanceof the required particle size in the case of dispersion and by the useof surfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Upon formulation, solutions will be administered in a manner compatiblewith the dosage formulation and in such amount as is therapeuticallyeffective. Formulations are easily administered in a variety of dosageforms, such as the type of injectable solutions described above, butdrug release capsules and the like can also be employed.

Suitable pharmaceutical compositions in accordance with the inventionwill generally include an amount of the coagulation-deficient TF admixedwith an acceptable pharmaceutical diluent or excipient, such as asterile aqueous solution, to give a range of final concentrations,depending on the intended use. The techniques of preparation aregenerally well known in the art as exemplified by Remington'sPharmaceutical Sciences, 16th Ed. Mack Publishing Company, 1980,incorporated herein by reference. It should be appreciated thatendotoxin contamination should be kept minimally at a safe level, forexample, less that 0.5 ng/mg protein. Moreover, for humanadministration, preparations should meet sterility, pyrogenicity,general safety and purity standards as required by FDA Office ofBiological Standards.

The therapeutically effective doses are readily determinable using ananimal model, as shown in the studies detailed herein. Experimentalanimals bearing solid tumors are frequently used to optimize appropriatetherapeutic doses prior to translating to a clinical environment. Suchmodels are known to be very reliable in predicting effective anti-cancerstrategies. For example, mice bearing solid tumors, such as used in theExamples, are widely used in pre-clinical testing. The inventors haveused such art-accepted mouse models to determine working ranges of tTFthat give beneficial anti-tumor effects with minimal toxicity.

In addition to the compounds formulated for parenteral administration,such as intravenous or intramuscular injection, other pharmaceuticallyacceptable forms are also contemplated, e.g., tablets or other solidsfor oral administration, time release capsules, liposomal forms and thelike. Other pharmaceutical formulations may also be used, dependent onthe condition to be treated. For example, topical formulations that areappropriate for treating pathological conditions such as dermatitis andpsoriasis; and ophthalmic formulations for diabetic retinopathy.

As described in detail herein, it is contemplated that certain benefitswill result from the manipulation of the coagulation-deficient TFconstructs to provide them with a longer in vivo half-life. Suchtechniques include, but are not limited to, manipulation or modificationof the TF molecule itself, and also conjugation of TF constructs toinert carriers, such as various protein or non-protein components,including immunoglobulins and Fc portions. Such compositions are hereintermed TF constructs with longer half-life. It will be understood thatlonger half-life is not coextensive with the pharmaceutical compositionsfor use in "slow release". Slow release formulations are generallydesigned to give a constant drug level over an extended period.Increasing the half-life of a drug, such as a TF construct in accordancewith the present invention, is intended to result in high plasma levelsupon administration, which levels are maintained for a longer time, butwhich levels generally decay depending on the pharmacokinetics of theconstruct. Although currently not preferred, slow release formulationsof the TF construct and combinations thereof are by no means excludedfrom use in the present invention.

C2. Therapeutic Kits

The present invention also provides therapeutic kits comprising the tTFconstructs described herein. Such kits will generally contain, insuitable container means, a pharmaceutically acceptable formulation ofat least one coagulation-deficient TF construct in accordance with theinvention. The kits may also contain other pharmaceutically acceptableformulations, such as those containing components to target the tTFconstructs; extra coagulation factors, particularly Factor VIIa;bispecific antibodies, T cells, or other functional components for usein, e.g., antigen induction; components for use in antigen suppression,such as a cyclosporin, if necessary; distinct anti-tumor site antibodiesor immunotoxins; and any one or more of a range of chemotherapeuticdrugs.

The kits may have a single container means that contains the tTF, withor without any additional components, or they may have distinctcontainer means for each desired agent. Kits comprising the separatecomponents necessary to make a bispecific coagulating ligand orimmunotoxin are also contemplated. Certain preferred kits of the presentinvention include a coagulation-deficient TF construct that is impairedin the ability to activate Factor VII, packaged in a kit for use incombination with the co-administration of exogenous Factor VIIa. In suchkits, the TF mutant and the Factor VIIa may be pre-complexed, either ina molar equivalent combination, or with one component in excess of theother; or each of the TF and Factor VIIa components of the kit may bemaintained separately within distinct containers prior to administrationto a patient. Other preferred kits include any coagulation-deficient TFin combination with a "classic" chemotherapeutic agent. This isexemplary of the considerations that are applicable to the preparationof all such TF kits and kit combinations in general.

When the components of the kit are provided in one or more liquidsolutions, the liquid solution is an aqueous solution, with a sterileaqueous solution being particularly preferred. However, the componentsof the kit may be provided as dried powder(s). When reagents orcomponents are provided as a dry powder, the powder can be reconstitutedby the addition of a suitable solvent. It is envisioned that the solventmay also be provided in another container means.

The container means of the kit will generally include at least one vial,test tube, flask, bottle, syringe or other container means, into whichthe tTF, and any other desired agent, may be placed and, preferably,suitably aliquoted. Where additional components are included, the kitwill also generally contain a second vial or other container into whichthese are placed, enabling the administration of separated designeddoses. The kits may also comprise a second/third container means forcontaining a sterile, pharmaceutically acceptable buffer or otherdiluent.

The kits may also contain a means by which to administer the tTF to ananimal or patient, e.g., one or more needles or syringes, or even an eyedropper, pipette, or other such like apparatus, from which theformulation may be injected into the animal or applied to a diseasedarea of the body. The kits of the present invention will also typicallyinclude a means for containing the vials, or such like, and othercomponent, in close confinement for commercial sale, such as, e.g.,injection or blow-molded plastic containers into which the desired vialsand other apparatus are placed and retained.

D. Treatment

D1. Prothrombotic Vessels

The compositions and methods provided by this invention are broadlyapplicable to the treatment of any disease, such as a benign ormalignant tumor, having as a component of the disease "prothromboticvessels". Such vasculature-associated diseases most particularly includesolid, malignant tumors, and also benign tumors, such as BPH. However,the treatment of diabetic retinopathy, vascular restenosis,arteriovenous malformations (AVM), meningioma, hemangioma, neovascularglaucoma and psoriasis; and also angiofibroma, arthritis,atherosclerotic plaques, corneal graft neovascularization, hemophilicjoints, hypertrophic scars, osler-weber syndrome, pyogenic granulomaretrolental fibroplasia, scleroderma, trachoma, vascular adhesions,synovitis, dermatitis and even endometriosis are certainly not excluded.

The present invention is based upon the use of TF constructs or TF incombination with other agents, wherein the TF construct or combinationhas sufficient thrombogenic activity to disturb the procoagulantenvironment within the specific disease-associated vessels, such asthose of a vascularized tumor, in the direction of thrombosis. Theenvironment in vessels in normal tissues is fibrinolytic, whereas thatin tumor vessels is procoagulant, i.e., predisposed towards thrombosis.The procoagulant changes in tumor vessels result in part from localrelease of the endothelial cell-activating cytokines, IL-1 and TNFα.IL-1 is secreted by most tumor cells and by activated macrophages. TNFαis secreted by host cells which have infiltrated into the tumor,including activated lymphocytes, macrophages, NK cells and LAK cells.

IL-1 and TNFα induce a variety of changes on vascular endothelium,including the upregulation of Tissue Factor, the down-regulation ofplasminogen activators and the upregulation of the inhibitor ofplasminogen activators, PAI-1 (Nawroth and Stern, 1986; Nawroth et al.,1988). These effects are further magnified by tumor derived factors(Murray et al., 1991; Ogawa et al., 1990), possibly VEGF. The collectiveresult of these and other changes is that the endothelium becomes betterable to support the formation of thrombi and less able to dissolvefibrin, producing a predisposition toward thrombosis.

Therefore, in light of the scientific phenomena described above, theinventors contemplate that when coagulation-deficient TFs areadministered, they have enough residual thrombogenic activity to tip thecoagulation cascade balance towards thrombosis in vessels that aregenerally prothrombotic in nature (FIG. 3). Although a mechanisticunderstanding of the scientific reasoning is not necessary in order topractice the present invention, it will be understood that the foregoingexplanation is one mechanism by which the invention may operate. Thismechanism is based less upon the specific localization of the TFs withinthe vessels of a vascularized tumor, as opposed to other vessels, but itis nonetheless surprising that an equal biodistribution of the TF, ifthis indeed occurs, can lead to an unequal effect on coagulation withindisease sites such as within solid tumors. Given that it is, naturally,an inherent property of the tumor to maintain a network of blood vesselsand to continue in the angiogenic process, it is evident that thetumor-associated blood vessels cannot be so predisposed towardsthrombosis that they spontaneously or readily support coagulation, assuch coagulation would necessarily result in the arrest of oxygen andnutrients to the tumor cells and would cause the tumor to self-destruct.Evidently, this does not occur.

It will be readily appreciated that the present invention hassignificant utility in the treatment of disease, such as vascularizedtumors, irrespective of an understanding of the mechanisms by whichspecific coagulation may be induced in disease-associated vessels.However, the inventors further reason that another mechanism underlyingthe possible surprising action of the TF constructs is that the TFsselectively bind to certain vascular endothelial cells in preference tothose in other tissues or sites of the body (FIG. 3). Accordingly,should tTF selectively bind to tumor vascular endothelium afterinjection, this would bring it into contact with a lipid surface andpromote the assembly of coagulation initiation complexes in the tumorvessels. Perhaps, because of the prothrombotic nature of tumor vessels,there is an increase in the local concentration of Factors VIIa, IXa,Xa, Tissue Factor pathway inhibitor (TFPI) or other molecules thatinteract with TF, thus encouraging the localization.

The methods of the present invention may be employed to test thelocalization of tTF by labeling tTF, injecting it into tumor-bearingmice, and determining whether it did indeed localize within tumorvessels. Although of scientific interest, conducting such studies arenot necessary to practice the present invention, given that theadministration of coagulation-deficient TF constructs advantageouslyresults in specific anti-tumor effects irrespective of the precisemechanism of action that underlies this phenomenon.

The present uses of coagulation-deficient TF molecules for promotingcoagulation in prothrombotic blood vessels are distinct to the previoususes proposed for TF constructs, such as tTF in combination with FactorVIIa. tTF and Factor VIIa have been proposed for combined use in thetreatment of bleeding disorders, such as hemophilia (U.S. Pat. Nos.5,374,617; 5,504,064; and 5,504,067). U.S. Pat. Nos. 5,346,991 and5,589,363 also describe the use of K165A and K166A TF mutants ininhibiting coagulation in the treatment of myocardial infarction, andprovide recombinant DNA sequences and vectors for their production.

It will be instantly appreciated that the targets of the previousmethodology are in direct contrast to the prothrombotic blood vesselstargeted by the present invention. The "prothrombotic" blood vessels arein a dynamic state that pre-disposes them to coagulation, but in whichcoagulation does not occur in the natural environment. This isexemplified by the blood vessels within a vascularized tumor beingcategorized as prothrombotic, but with the tumor maintaining asufficient blood supply throughout necessary to support the maintenanceand outgrowth of the tumor. In contrast, the target sites within anindividual with a bleeding disorder, are by their very naturesignificantly deficient in their ability to support coagulation. Thecombined tTF and Factor VIIa methodology intended primarily for use inhemophiliacs has also been proposed for use in conjunction with thecontrol of postoperative bleeding or severe trauma in which an externalinsult has prevented the necessary coagulation process from beingeffective. This again is unlike the intent of the present invention.

The most important use of the present invention is believed to be inconnection with the treatment of vascularized, malignant tumors.However, in addition to the various diseases and disorders listed above,the invention is also contemplated for use in the therapy of otherbenign growths. A particular example of such is benign prostatichyperplasia (BPH), which may be treated in accordance with theparticular doses and treatment regimens set forth below. The treatmentof BPH may also be combined with other treatments currently practiced inthe art. For example, targeting of immunotoxins to markers localizedwithin BPH, such as PSA, is certainly contemplated.

D2. Cancer and Treatment

The tTF localization and specific coagulation of the invention is mostpreferably exploited for therapeutic uses of tTF in the treatment ofcancers and tumors. These uses may employ tTF alone or in combinationwith chemotherapeutic agents and/or immunotoxins or coaguligands. Thecompositions and methods provided by this invention are broadlyapplicable to the treatment of any malignant tumor having a vascularcomponent. Typical vascularized tumors are the solid tumors,particularly carcinomas, which require a vascular component for theprovision of oxygen and nutrients. Exemplary solid tumors that may betreated using the invention include, but are not limited to, carcinomasof the lung, breast, ovary, stomach, pancreas, larynx, esophagus,testes, liver, parotid, biliary tract, colon, rectum, cervix, uterus,endometrium, kidney, bladder, prostate, thyroid, squamous cellcarcinomas, adenocarcinomas, small cell carcinomas, melanomas, gliomas,neuroblastomas, and the like.

The present invention is contemplated for use in the treatment of anypatient that presents with a solid tumor. However, in that the presentinvention is particularly successful in the treatment of solid tumors ofmoderate or large sizes, patients in these categories are likely toreceive more significant benefits from treatments in accordance with themethods and compositions provided herein. In general, the invention canbe used to treat tumors of about 0.3-0.5 cm and upwards, although it isa better use of the invention to treat tumors of greater than 0.5 cm insize. From the studies already conducted in acceptable animal models, itis believed that tumors of about 1.0 or about 1.2 cm represent the sizeof solid tumors that are most effectively attacked by the TF constructsof the present invention. Therefore, patients presenting with tumors ofbetween about 1.0 and about 2.0 cm in size will be in the preferredtreatment group of patients in connection with the present TF therapies,although tumors up to and including the largest tumors found in humansmay also be treated.

Although the present invention is not generally intended as apreventative or prophylactic treatment, use of the invention iscertainly not confined to the treatment of patients having tumors ofmoderate or large sizes. There are many reasons underlying this aspectof the breadth of the invention. For example, a patient presenting witha primary tumor of moderate size or above may also have various othermetastatic tumors that are considered to be small-sized or even in theearlier stages of metastatic tumor seeding. Given that the TF constructsand combinations of the invention are generally administered into thesystemic circulation of a patient, they will naturally have effects onthe secondary, smaller and metastatic tumors, although this may not bethe primary intent of the treatment. Furthermore, even in situationswhere the tumor mass as a whole is a single small tumor, certainbeneficial anti-tumor effects will result from the use of the presenttreatments.

The guidance provided above regarding the most suitable patients for usein connection with the present invention is intended as teaching thatcertain patient's profiles may assist with the selection of patientsthat may be treated by the present invention, or that may, perhaps, bebetter treated using other anti-cancer treatment strategies.Nonetheless, the fact that a preferred or otherwise more effectivetreatment is perceived to exist in connection with a certain category ofpatients, does not in any way negate the basic utility of the presentinvention in connection with the treatments of all patients having avascularized tumor. A further consideration is the fact that the initialassault on a tumor, as provided by the TF therapy of the presentinvention, may be small in any measurable and immediate effects, but maypredispose the tumor to further therapeutic treatments such that thesubsequent treatment results in an overall synergistic effect or evenleads to total remission or cure.

It is not believed that any particular type of tumor should be excludedfrom treatments using the present invention. As the intent of thetherapy is to coagulate the tumor vasculature, and as the vasculature issubstantially or entirely the same in all solid tumors, it will beunderstood that the present methodology is widely or entirely applicableto the treatment of all solid tumors, irrespective of the particularphenotype or genotype of the tumor cells themselves. However, the typeof tumor cells may be relevant to the use of the invention incombination with secondary therapeutic agents, particularly anti-tumorcell immunotoxins and/or coaguligands.

Those of ordinary skill in the art will understand that certain types oftumors may be more amenable to the induction of thrombosis and necrosisusing the present invention. The phenomena is observed in experimentalanimals, and may occur in human treatments. For example, it is knownthat the HT29 model tumor system is relatively difficult to coagulate;whereas the C1300 tumor model is generally more amenable to theinduction of thrombosis and subsequent necrosis. Such considerationswill be taken into account in conducting both the pre-clinical studiesin experimental animals and in optimizing the doses for use in treatingany particular patient or group of patients.

As detailed above in the discussions concerning the in vivo quantitativestudies, there are realistic objectives that may be used as a guidelinein connection with pre-clinical testing before proceeding to clinicaltreatment. However, this is more a matter of cost-effectiveness thanoverall usefulness, and is a mechanism for selecting the mostadvantageous compounds and doses. In regard to their basic utility, anyconstruct or combination thereof that results in any consistentdetectable thrombosis and anti-tumor effects will still define a usefulinvention. Thrombotic and necrotic effects should be observed in betweenabout 10% and about 40-50% of the tumor blood vessels and tumor tissues,upwards to between about 50% and about 99% of such effects beingobserved. It will also be understood that even in such circumstanceswhere the anti-tumor effects of the TF construct and combinations aretowards the low end of this range, it may be that this therapy is stillequally or even more effective than all other known therapies in thecontext of the particular tumor targets. It is unfortunately evident toa clinician that certain tumors cannot be effectively treated in theintermediate or long term, but that does not negate the usefulness ofthe present therapy, particularly where it is about as effective as theother strategies generally proposed.

In designing appropriate doses of the coagulation-deficient TFconstructs and combinations, one may readily extrapolate from the animalstudies described herein in order to arrive at appropriate doses forclinical administration. To achieve this conversion, one would accountfor the mass of the agents administered per unit mass of theexperimental animal, and yet account for the differences in the bodysurface area between the experimental animal and the human patient. Allsuch calculations are well known and routine to those of ordinary skillin the art. For example, in taking the successful dose of 16 μg permouse (total body weight of about 20 g), and applying the calculationoutlined above, the equivalent dose for use in a human patient would beabout 2 mg. Accordingly, using this information, the inventorscontemplate that useful doses of coagulation-deficient TF for use inhuman administration would be between about 0.2 milligrams and about 200milligrams of the TF construct per patient. Notwithstanding this statedrange, it will be understood that, given the parameters and detailedguidance presented above, further variations in the active or optimalranges would still be encompassed within the present invention.

The doses contemplated will therefore generally be between about 0.2 mgand about 180 milligrams; between 0.5 and about 160 milligrams; between1 and about 150 milligrams; between about 5 and about 125 milligrams;between about 10 and about 100 milligrams; between about 15 and about 80milligrams; between about 20 and about 65 milligrams; between about 30and about 50 milligrams; about 40 milligrams; or in any particular rangeusing any of the foregoing recited exemplary doses or any valueintermediate between the particular stated ranges. Although doses in andaround about 1 milligram, 2 milligrams, 3 milligrams, 4 milligrams andabout 5 milligrams are currently preferred, it will be understood thatlower doses may be more appropriate in combination with other agents,and that high doses can still be tolerated, particularly given the factthat the TF agents for use in the invention are not themselves cytotoxicand even if certain adverse side effects do occur, this should notnecessarily result in coagulation that cannot be counteracted by normalhomeostatic mechanisms, which is believed to lessen the chances ofsignificant toxicity to healthy tissues.

The intention of the therapeutic regimens of the present invention isgenerally to produce the maximum anti-tumor effects whilst still keepingthe dose below the levels associated with unacceptable toxicity. Inaddition to varying the dose itself, the administration regimen can alsobe adapted to optimize the treatment strategy. A currently preferredtreatment strategy is to administer between about 0.2 milligrams andabout 200 milligrams of the TF construct or combination thereof about 3times within about a 7 day period. For example, doses would be given onabout day 1, day 3 or 4 and day 6 or 7. In administering the particulardoses themselves, one would preferably provide a pharmaceuticallyacceptable composition to the patient systemically. Intravenousinjection is generally preferred, and the most preferred method is toemploy a continuous infusion over a time period of about 1 or 2 hours orso. Although it is not required to determine such parameters prior totreatment using the present invention, it should be noted that thestudies detailed herein result in at least some thrombosis beingobserved specifically in the blood vessels of a solid tumor within about30 minutes of injection, and that the tumor cells themselves begin todie within about 3 to 4 hours. Widespread tumor necrosis is generallyobserved in the next about 24 hours, up to and including greater than90% necrosis being observed.

E. Combination Therapies

The methods of the present invention may be combined with any othermethods generally employed in the treatment of the particular disease ordisorder that the patient exhibits. For example, in connection with thetreatment of solid tumors, the methods of the present invention may beused in combination with classical approaches, such as surgery,radiotherapy and the like. So long as a particular therapeutic approachis not known to be detrimental in itself, or counteracts theeffectiveness of the TF therapy, its combination with the presentinvention is contemplated. When one or more agents are used incombination with the TF therapy, there is no requirement for thecombined results to be additive of the effects observed when eachtreatment is conducted separately, although this is evidently desirable,and there is no particular requirement for the combined treatment toexhibit synergistic effects, although this is certainly possible andadvantageous.

In terms of surgery, any surgical intervention may be practiced incombination with the present invention. In connection with radiotherapy,any mechanism for inducing DNA damage locally within tumor cells iscontemplated, such as γ-irradiation, X-rays, UV-irradiation, microwavesand even electronic emissions and the like. The directed delivery ofradioisotopes to tumor cells is also contemplated, and this may be usedin connection with a targeting antibody or other targeting means.Cytokine therapy also has proven to be an effective partner for combinedtherapeutic regimens. Various cytokines may be employed in such combinedapproaches. Examples of cytokines include IL-1α IL-1β, IL-2, IL-3, IL-4,IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, TGF-β, GM-CSF,M-CSF, G-CSF, TNFα, TNFβ, LAF, TCGF, BCGF, TRF, BAF, BDG, MP, LIF, OSM,TMF, PDGF, IFN-α, IFN-β, IFN-γ. Cytokines are administered according tostandard regimens, consistent with clinical indications such as thecondition of the patient and relative toxicity of the cytokine.

E1. Chemotherapeutic Combinations and Treatment

In certain embodiments, the present invention shows that the anti-tumoractivity of tTF is enhanced when administered in combination with achemotherapeutic agent. The mechanisms by which the drugs enhance theanti-tumor activity of tTF have not been precisely defined, but theinventors believe that the drug kills proliferating tumor cells creatingnecrotic areas that cause phagocytic cells to infiltrate the tumor.IL-1, TNFα and other cytokines released by the infiltrating cells thenactivate the tumor vascular endothelium making it better able to supportcoagulation by tTF, a generally weak thrombogen. The drug thus enhancesthe thrombotic action of tTF.

Another possibility for the enhanced actions of TF and anti-cancer drugsis that tTF induces the formation of thrombi in tumor vessels, therebytrapping drug within the tumor. While drug is cleared from the rest ofthe body, it remains within the tumor. The tumor cells are thus exposedto a higher concentration of drug for a longer period of time. Thisentrapment of drug within the tumor may also make it possible to reducethe dose of drug, making the treatment safer as well as more effective.

Irrespective of the mechanisms by which the enhanced tumor destructionis achieved, the combined treatment aspects of the present inventionhave evident utility in the effective treatment of disease. To use thepresent invention in combination with the administration of achemotherapeutic agent, one would simply administer to an animal acoagulation-deficient TF construct in combination with thechemotherapeutic agent in a manner effective to result in their combinedanti-tumor actions within the animal. These agents would therefore beprovided in an amount effective and for a period of time effective toresult in their combined presence within the tumor vasculature and theircombined actions in the tumor environment. To achieve this goal, the TFand chemotherapeutic agents may be administered to the animalsimultaneously, either in a single composition or as two distinctcompositions using different administration routes.

Alternatively, the TF treatment may precede or follow thechemotherapeutic agent treatment by intervals ranging from minutes toweeks. In embodiments where the chemotherapeutic factor and TF areapplied separately to the animal, one would generally ensure that asignificant period of time did not expire between the time of eachdelivery, such that the chemotherapeutic agent and TF composition wouldstill be able to exert an advantageously combined effect on the tumor.In such instances, it is contemplated that one would contact the tumorwith both agents within about 5 minutes to about one week of each otherand, more preferably, within about 12-72 hours of each other, with adelay time of only about 12-48 hours being most preferred. In somesituations, it may be desirable to extend the time period for treatmentsignificantly, where several days (2, 3, 4, 5, 6 or 7) or even severalweeks (1, 2, 3, 4, 5, 6, 7 or 8) lapse between the respectiveadministrations. It also is conceivable that more than oneadministrations of either the TF or the chemotherapeutic agent will bedesired. To achieve tumor regression, both agents are delivered in acombined amount effective to inhibit its growth, irrespective of thetimes for administration.

A variety of chemotherapeutic agents are intended to be of use in thecombined treatment methods disclosed herein. Chemotherapeutic agentscontemplated as exemplary include, e.g., etoposide (VP-16), adriamycin,5-fluorouracil (5FU), camptothecin, actinomycin-D, mitomycin C,cisplatin (CDDP) and even hydrogen peroxide. In certain embodiments, theuse of etoposide has already been shown to be effective in regression oftumor in size when administered in combination with the tTF compositionsof the present invention.

As will be understood by those of ordinary skill in the art, theappropriate doses of chemotherapeutic agents will be generally aroundthose already employed in clinical therapies wherein thechemotherapeutics are administered alone or in combination with otherchemotherapeutics. By way of example only, agents such as cisplatin, andother DNA alkylating may be used. Cisplatin has been widely used totreat cancer, with efficacious doses used in clinical applications of 20mg/m² for 5 days every three weeks for a total of three courses.Cisplatin is not absorbed orally and must therefore be delivered viainjection intravenously, subcutaneously, intratumorally orintraperitoneally.

Further useful agents include compounds that interfere with DNAreplication, mitosis and chromosomal segregation. Such chemotherapeuticcompounds include adriamycin, also known as doxorubicin, etoposide,verapamil, podophyllotoxin, and the like. Widely used in a clinicalsetting for the treatment of neoplasms, these compounds are administeredthrough bolus injections intravenously at doses ranging from 25-75 mg/m²at 21 day intervals for adriamycin, to 35-50 mg/m² for etoposideintravenously or double the intravenous dose orally.

Agents that disrupt the synthesis and fidelity of polynucleotideprecursors may also be used. Particularly useful are agents that haveundergone extensive testing and are readily available. As such, agentssuch as 5-fluorouracil (5-FU) are preferentially used by neoplastictissue, making this agent particularly useful for targeting toneoplastic cells. Although quite toxic, 5-FU, is applicable in a widerange of carriers, including topical, however intravenous administrationwith doses ranging from 3 to 15 mg/kg/day being commonly used.

Exemplary chemotherapeutic agents that are useful in connection withcombined therapy are listed in Table II. Each of the agents listedtherein are exemplary and by no means limiting. The skilled artisan isdirected to "Remington's Pharmaceutical Sciences" 15th Edition, chapter33, in particular pages 624-652. Some variation in dosage willnecessarily occur depending on the condition of the subject beingtreated. The person responsible for administration will, in any event,determine the appropriate dose for the individual subject. Moreover, forhuman administration, preparations should meet sterility, pyrogenicity,general safety and purity standards as required by FDA Office ofBiologics standards.

                                      TABLE II                                    __________________________________________________________________________    CHEMOTHERAPEUTIC AGENTS USEFUL IN NEOPLASTIC DISEASE                                             NONPROPRIETARY                                                                NAMES                                                      CLASS   TYPE OF AGENT                                                                            (OTHER NAMES)                                                                             DISEASE                                        __________________________________________________________________________    Alkylating Agents                                                                     Nitrogen Mustards                                                                        Mechlorethamine (HN.sub.2)                                                                Hodgkin's disease, non-Hodgkin's                                              lymphomas                                                         Cyclophosphamide                                                                          Acute and chronic lymphocytic                                     Ifosfamide  leukemias, Hodgkin's disease, non-                                            Hodgkin's lymphomas, multiple                                                 myeloma, neuroblastoma, breast,                                               ovary, lung, Wilms' tumor, cervix,                                            testis, soft-tissue sarcomas                                      Melphalan (t-sarcolysin)                                                                  Multiple myeloma, breast, ovary                                   Chlorambucil                                                                              Chronic lymphocytic leukemia,                                                 primary macroglobulinemia, Hodgkin's                                          disease, non-Hodgkin's lymphomas                       Ethyleneimenes and                                                                       Hexamethylmelamine                                                                        Ovary                                                  Methylmelamines                                                                          Thiotepa    Bladder, breast, ovary                                 Alkyl Sulfonates                                                                         Busulfan    Chronic granulocytic leukemia                          Nitrosoureas                                                                             Carmustine (BCNU)                                                                         Hodgkin's disease, non-Hodgkin's                                              lymphomas, primary brain tumors,                                              multiple myeloma, malignant                                                   melanoma                                                          Lomustine (CCNU)                                                                          Hodgkin's disease, non-Hodgkin's                                              lymphomas, primary brain tumors,                                              small-cell lung                                                   Semustine (methyl-CCNU)                                                                   Primary brain tumors, stomach, colon                              Streptozocin                                                                              Malignant pancreatic insulinoma,                                  (streptozotocin)                                                                          malignant carcinoid                                    Triazines  Dacarbazine (DTIC;                                                                        Malignant melanoma, Hodgkin's                                     dimethyltriazenoimidaz                                                                    disease, soft-tissue sarcomas                                     olecarboxamide)                                            Antimetabolites                                                                       Folic Acid Analogs                                                                       Methotrexate                                                                              Acute lymphocytic leukemia,                                       (amethopterin)                                                                            choriocarcinoma, mycosis fungoides,                                           breast, head and neck, lung,                                                  osteogenic sarcoma                                     Pyrimidine Analogs                                                                       Fluouracil (5-fluorouracil;                                                               Breast, colon, stomach, pancreas,                                 5-FU)       ovary, head and neck, urinary bladder,                            Floxuridine (fluorode-                                                                    premalignant skin lesions (topical)                               oxyuridine; FUdR)                                                             Cytarabine (cytosine                                                                      Acute granulocytic and acute                                      arabinoside)                                                                              lymphocytic leukemias                                  Purine Analogs and                                                                       Mercaptopurine                                                                            Acute lymphocytic, acute                               Related Inhibitors                                                                       (6-mercaptopurine;                                                                        granulocytic and chronic granulocytic                             6-MP)       leukemias                                                         Thioguanine Acute granulocytic, acute                                         (6-thioguanine; TG)                                                                       lymphocytic and chronic granulocytic                                          leukemias                                                         Pentostatin Hairy cell leukemia, mycosis                                      (2-deoxycoformycin)                                                                       fungoides, chronic lymphocytic                                                leukemia                                       Natural Products                                                                      Vinca Alkaloids                                                                          Vinblastine (VLB)                                                                         Hodgkin's disease, non-Hodgkin's                                              lymphomas, breast, testis                                         Vincristine Acute lymphocytic leukemia,                                                   neuroblastoma, Wilms' tumor,                                                  rhabdomyosarcoma, Hodgkin's                                                   disease, non-Hodgkin's lymphomas,                                             small-cell lung                                        Epipodophyllotoxins                                                                      Etoposide   Testis, small-cell lung and other lung,                           Tertiposide breast, Hodgkin's disease, non-                                               Hodgkin's lymphomas, acute                                                    granulocytic leukemia, Kaposi's                                               sarcoma                                                Antibiotics                                                                              Dactinomycin                                                                              Choriocarcinoma, Wilms' tumor,                                    (actinomycin D)                                                                           rhabdomyosarcoma, testis, Kaposi's                                            sarcoma                                                           Daunorubicin                                                                              Acute granulocytic and acute                                      (daunomycin;                                                                              lymphocytic leukemias                                             rubidomycin)                                                                  Doxorubicin Soft-tissue, osteogenic and other                                             sarcomas; Hodgkin's disease, non-                                             Hodgkin's lymphomas, acute                                                    leukemias, breast, genitourinary,                                             thyroid, lung, stomach, neuroblastoma                             Bleomycin   Testis, head and neck, skin,                                                  esophagus, lung and genitourinary                                             tract; Hodgkin's disease, non-                                                Hodgkin's lymphomas                                               Plicamycin (mithramycin)                                                                  Testis, malignant hypercalcemia                                   Mitomycin (mitomycin C)                                                                   Stomach, cervix, colon, breast,                                               pancreas, bladder, head and neck                       Enzymes    L-Asparaginase                                                                            Acute lymphocytic leukemia                             Biological Response                                                                      Interferon alfa                                                                           Hairy cell leukemia., Kaposi's                         Modifiers              sarcoma, melanoma, carcinoid, renal                                           cell, ovary, bladder, non-Hodgkin's                                           lymphomas, mycosis fungoides,                                                 multiple myeloma, chronic                                                     granulocytic leukemia                          Miscellaneous                                                                         Platinum Coordination                                                                    Cisplatin (cis-DDP)                                                                       Testis, ovary, bladder, head and neck,         Agents  Complexes  Carboplatin lung, thyroid, cervix, endometrium,                                           neuroblastoma, osteogenic sarcoma                      Anthracenedione                                                                          Mitoxantrone                                                                              Acute granulocytic leukemia, breast                    Substituted Urea                                                                         Hydroxyurea Chronic granulocytic leukemia,                                                polycythemia vera, essental                                                   thrombocytosis, malignant melanoma                     Methyl Hydrazine                                                                         Procarbazine                                                                              Hodgkin's disease                                      Derivative (N-methylhydrazine,                                                           MIH)                                                               Adrenocortical                                                                           Mitotane (o,p'-DDD)                                                                       Adrenal cortex                                         Suppressant                                                                              Aminoglutethimide                                                                         Breast                                         Hormones and                                                                          Adrenocorticosteroids                                                                    Prednisone (several other                                                                 Acute and chronic lymphocytic                  Antagonists        equivalent  leukemias, non-Hodgkin's lymphomas,                               preparations available)                                                                   Hodgkin's disease, breast                              Progestins Hydroxyprogesterone                                                                       Endometrium, breast                                               caproate                                                                      Medroxyprogesterone                                                           acetate                                                                       Megestrol acetate                                                  Estrogens  Diethylstilbestrol                                                                        Breast, prostate                                                  Ethinyl estradiol (other                                                      preparations available)                                            Antiestrogen                                                                             Tamoxifen   Breast                                                 Androgens  Testosterone propionate                                                                   Breast                                                            Fluoxymesterone (other                                                        preparations available)                                            Antiandrogen                                                                             Flutamide   Prostate                                               Gonadotropin-releasing                                                                   Leuprolide  Prostate                                               hormone analog                                                        __________________________________________________________________________

E2. Immunotoxin and Coaguligand Combinations and Therapy

Any one or more of the coagulation-deficient TF constructs of theinvention may be used in combination with immunotoxins (ITs) and/orcoaguligands in which the targeting portion thereof (e.g., antibody orligand) is directed to a relatively specific marker of the tumor cells,tumor vasculature or tumor stroma. In common with the chemotherapeuticagents discussed above, it is possible that the use of acoagulation-deficient TF construct in combination with a targeted toxicagent (IT) or coagulant (coaguligand) will result in the distinct agentsbeing directed against different targets within the tumor environment.This should result in additive, markedly greater than additive or evensynergistic results.

In connection with the preparation and use of exemplary immunotoxins andcoaguligands, the following patent application disclosures arespecifically incorporated herein by reference for the purposes of evenfurther supplementing the present teachings: U.S. Ser. Nos. 07/846,349;08/295,868; 08/205,330; 08/350,212; 08/273,567; and 08/482,369.

At least one binding region of the second agents employed in combinationwith the tTF constructs of the present invention will be a componentthat is capable of delivering a toxin or coagulating agent to the tumorregion, i.e., capable of localizing within a tumor site. As somewhatwider distribution of a coagulating agent will not be associated withsevere side effects, there is a less stringent requirement imposed onthe targeting element of coaguligands than with immunotoxins. Eithertargeting agent may be directed to components of tumor cells; componentsof tumor vasculature; components that bind to, or are generallyassociated with, tumor cells; components that bind to, or are generallyassociated with, tumor vasculature; components of the tumorextracellular matrix or stroma or those bound therein; and even celltypes found within the tumor vasculature.

With coaguligands, the burden of very stringent targeting, e.g., asimposed when using immunotoxins, is lessened. Therefore, to achievespecific targeting means that coagulation is promoted in the tumorvasculature relative to the vasculature in non-tumor sites. Thus,specific targeting of a coaguligand is a functional term rather than apurely physical term relating to the biodistribution properties of thetargeting agent, and it is not unlikely that useful targets may be notbe entirely tumor-restricted, and that targeting ligands which areeffective to promote tumor-specific coagulation may nevertheless befound at other sites of the body following administration.

i. Tumor Cell Targets

The malignant cells that make up the tumor may be targeted using aligand or bispecific ligand that has a region capable of binding to arelatively specific marker of the tumor cell. Toxins kill the tumorcells and, in that binding to tumor cells will localize an associatedcoagulating agent to the tumor, specific coagulation will be achieved.

Many so-called "tumor antigens" have been described, any one which couldbe employed as a target in connection with the combined aspects of thepresent invention. A large number of exemplary solid tumor-associatedantigens are listed herein below. The preparation and use of antibodiesagainst such antigens is well within the skill of the art, and exemplaryantibodies include from gynecological tumor sites: OC 125; OC 133; OMI;Mo v1; Mo v2; 3C2; 4C7; ID₃ ; DU-PAN-2; F 36/22; 4F₇ /7A₁₀ ; OV-TL3;B72.3; DF₃ ; 2C₈ /2F₇ ; MF 116; Mov18; CEA 11H5; CA 19-9 (1116NS 19-9);H17-E2; 791T/36; NDOG₂ ; H317; 4D5, 3H4, 7C2, 6E9, 2C4, 7F3, 2H11, 3E8,5B8, 7D3, SB8; HMFG2; 3.14.A3; from breast tumor sites: DF3; NCRC-11;3C6F9; MBE6; CLNH5; MAC 40/43; EMA; HMFG1 HFMG2; 3.15.C3; M3, M8, M24;M18; 67-D-11; D547Sp, D75P3, H222; Anti-EGF; LR-3; TA1; H59; 10-3D-2;HmAB1,2; MBR 1,2,3; 24.17.1; 24.17.2 (3E1.2); F36/22.M7/105; C11, G3,H7; B6.2; B1.1; Cam 17.1; SM3; SM4; C-Mul (566); 4D5 3H4, 7C2, 6E9, 2C4,7F3, 2H11, 3E8, 5B8, 7D3, 5B8; OC 125; MO v2; DU-PAN-2; 4F₇ /7A₁₀ ; DF₃; B72.3; cccccCEA 11; H17-E2; 3.14.A3; FO23C5; from colorectal tumorsites: B72.3; (17-1A) 1083-17-1A; CO17-1A; ZCE-025; AB2; HT-29-15;250-30.6; 44X14; A7; GA73.3; 791T/36; 28A32; 28.19.8; X MMCO-791;DU-PAN-2; ID₃ ; CEA 11-H5; 2C₈ /2F₇ ; CA-19-9 (1116NS 19-9); PR5C5;PR4D2; PR4D1; from melanoma sites 4.1; 8.22 M₁₇ ; 96.5; 118.1, 133.2,(113.2); L₁, L₁₀, R₁₀ (R₁₉); I₁₂ ;K₅ ; 6.1;R24; 5.1; 225.28S; 465.12S;9.2.27;F11;376.96S; 465.12S; 15.75; 15.95;Mel-14;Mel-12;Me3-TB7;225.28SD; 763.24TS; 705F6; 436910; M148;from gastrointestinal tumors:ID3;DU-PAN-2;OV-TL3;B72.3;CEA 11-H5; 3.14.A3;C COLI; CA-19-9 (1116NS19-9) and CA50;OC125;from lung tumors: 4D5 3H4, 7C2, 6E9, 2C4, 7F3,2H11, 3E8, 5B8, 7D3, SB8;MO v2;B72.3;DU-PAN-2;CEA 11-H5;MUC 8-22;MUC2-63; MUC 2-39;MUC 7-39;and from miscellaneous tumors: PAb 240;PAb246;PAb 1801; ERIC-1;M148;FMH25; 6.1;CA1; 3F8; 4F₇ /7A₁₀ ; 2C₈ /2F₇ ;CEA11-H5.

Another means of defining a targetable tumor is in terms of thecharacteristics of a tumor cell itself, rather than describing thebiochemical properties of an antigen expressed by the cell. Accordingly,the skilled artisan is referred to the ATCC catalogue for the purpose ofexemplifying human tumor cell lines that are publicly available (fromATCC Catalogue). Exemplary cell lines include J82;RT4;ScaBER;T24;TCCSUP;5637;SK-N-MC; SK-N-SH; SW 1088;SW 1783;U-87 MG; U-118 MG;U-138 MG; U-373 MG; Y79;BT-20;BT-474; MCF7;MDA-MB-134-VI;MDA-MD-157;MDA-MB-175-VII; MDA-MB-361;SK-BR-3;C-33 A;HT-3;ME-180;MS751;SiHa; JEG-3;Caco-2;HT-29;SK-CO-1;HuTu 80;A-253;FaDu;A-498; A-704;Caki-1;Caki-2;SK-NEP-1;SW839;SK-HEP-1;A-427;Calu-1;Calu-3;Calu-6; SK-LU-1;SK-MES-1;SW900;EB1;EB2;P3HR-1;HT-144;Malme-3M; RPMI-7951;SK-MEL-1;SK-MEL-2;SK-MEL-3;SK-MEL-5;SK-MEL-24;SK-MEL-28;SK-MEL-31;Caov-3;Caov-4;SK-OV-3;SW 626;Capan-1;Capan-2;DU 145;A-204;Saos-2;SK-ES-1;SK-LMS-1; SW 684;SW 872;SW 982;SW 1353;U-2 OS; Malme-3;KATO III;Cate-1B; Tera-1;Tera-2; SW579;AN3 CA; HEC-1-A; HEC-1-B;SK-UT-1;SK-UT-1B; SW 954;SW 962;NCI-H69;NCI-H128;BT-483;BT-549;DU4475;HBL-100;Hs 578Bst; Hs 578T;MDA-MB-330;MDA-MB-415; MDA-MB-435S;MDA-MB-436;MDA-MB-453;MDA-MB-468;T-47D; Hs 766T; Hs 746T; Hs 695T; Hs683;Hs 294T; Hs 602;JAR; Hs 445;Hs 700T; H4;Hs 696;Hs 913T; Hs 729;FHs738Lu; FHs 173We; FHs 738B1;NIH:0VCAR-3;Hs 67;RD-ES; ChaGo K-1;WERI-Rb-1; NCI-H446;NCI-H209;NCI-H146;NCI-H441;NCI-H82;1H9;NCI-H460;NCI-H596; NCI-H676B;NCI-H345;NCI-H820;NCI-H520;NCI-H661;NCI-H510A; D283 Med; Daoy; D341 Med;AML-193 and MV4-11.

One may consult the ATCC Catalogue of any subsequent year to identifyother appropriate cell lines. Also, if a particular cell type isdesired, the means for obtaining such cells, and/or their instantlyavailable source, will be known to those of skill in the particular art.An analysis of the scientific literature will thus readily reveal anappropriate choice of cell for any tumor cell type desired to betargeted.

(a) Anti-Tumor Cell Antibodies

A straightforward means of recognizing a tumor antigen target is throughthe use of an antibody that has binding affinity for the particularantigen. An extensive number of antibodies are known that are directedagainst solid tumor antigens. Certain useful anti-tumor antibodies arelisted above. However, as will be instantly known to those of skill inthe art, certain of the antibodies listed will not have the appropriatebiochemical properties, or may not be of sufficient tumor specificity,to be of use therapeutically. An example is MUC8-22 that recognizes acytoplasmic antigen. Antibodies such as these will generally be of useonly in investigational embodiments, such as in model systems orscreening assays.

Generally speaking, antibodies for use in these aspects of the presentinvention will preferably recognize antigens that are accessible on thecell-surface and that are preferentially, or specifically, expressed bytumor cells. Such antibodies will also preferably exhibit properties ofhigh affinity, such as exhibiting a K_(d) of <200 nM, and preferably, of<100 nM, and will not show significant reactivity with life-sustainingnormal tissues, such as one or more tissues selected from heart, kidney,brain, liver, bone marrow, colon, breast, prostate, thyroid, gallbladder, lung, adrenals, muscle, nerve fibers, pancreas, skin, or otherlife-sustaining organ or tissue in the human body. The "life-sustaining"tissues that are the most important for the purposes of the presentinvention, from the standpoint of low reactivity, include heart, kidney,central and peripheral nervous system tissues and liver. The term"significant reactivity", as used herein, refers to an antibody orantibody fragment, that, when applied to the particular tissue underconditions suitable for immunohistochemistry, will elicit either nostaining or negligible staining with only a few positive cells scatteredamong a field of mostly negative cells.

Particularly promising antibodies contemplated for use in the presentinvention are those having high selectivity for the solid tumor. Forexample, antibodies binding to TAG 72 and the HER-2 proto-oncogeneprotein, which are selectively found on the surfaces of many breast,lung and colorectal cancers (Thor et al., 1986;Colcher et al.,1987;Shepard et al., 1991); MOv18 and OV-TL3 and antibodies that bind tothe milk mucin core protein and human milk fat globule (Miotti et al.,1985;Burchell et al., 1983); and the antibody 9.2.27 that binds to thehigh M_(r) melanoma antigens (Reisfeld et al., 1982). Further usefulantibodies are those against the folate-binding protein, which is knownto be homogeneously expressed in almost all ovarian carcinomas; thoseagainst the erb family of oncogenes that are over-expressed in squamouscell carcinomas and the majority of gliomas; and other antibodies knownto be the subject of ongoing pre-clinical and clinical evaluation.

The antibodies B3, KSI/4, CC49, 260F9, XMMCO-791, D612 and SM3 arebelieved to be particularly suitable for use in clinical embodiments,following the standard pre-clinical testing routinely practiced in theart. B3 (U.S. Pat. No. 5,242,813;Brinkmann et al., 1991) has ATCCAccession No. HB 10573;KSI/4 can be made as described in U.S. Pat. No.4,975,369;and D612 (U.S. Pat. No. 5,183,756) has ATCC Accession No. HB9796.

Another means of defining a tumor-associated target is in terms of thecharacteristics of the tumor cell, rather than describing thebiochemical properties of an antigen expressed by the cell. Accordingly,the inventors contemplate that any antibody that preferentially binds toa tumor cell may be used as the targeting component of an immunotoxin orcoaguligand. The preferential tumor cell binding is again based upon theantibody exhibiting high affinity for the tumor cell and not havingsignificant reactivity with life-sustaining normal cells or tissues, asdefined above.

The invention also provides several means for generating an antibody foruse in the targeted coagulation methods described herein. To generate atumor cell-specific antibody, one would immunize an animal with acomposition comprising a tumor cell antigen and, as described more fullyin below, select a resultant antibody with appropriate specificity. Theimmunizing composition may contain a purified, or partially purified,preparation of any of the antigens in listed above; a composition, suchas a membrane preparation, enriched for any of the antigens in listedabove; any of the cells listed in listed above; or a mixture orpopulation of cells that include any of the cell types listed above.

Of course, regardless of the source of the antibody, in the practice ofthe invention in human treatment, one will prefer to ensure in advancethat the clinically-targeted tumor expresses the antigen ultimatelyselected. This is achieved by means of a fairly straightforward assay,involving antigenically testing a tumor tissue sample, for example, asurgical biopsy, or perhaps testing for circulating shed antigen. Thiscan readily be carried out in an immunological screening assay such asan ELISA (enzyme-linked immunosorbent assay), wherein the bindingaffinity of antibodies from a "bank" of hybridomas are tested forreactivity against the tumor. Antibodies demonstrating appropriate tumorselectivity and affinity are then selected for the preparation ofbispecific antibodies of the present invention.

Due to the well-known phenomenon of cross-reactivity, it is contemplatedthat useful antibodies may result from immunization protocols in whichthe antigens originally employed were derived from an animal, such as amouse or a primate, in addition to those in which the original antigenswere obtained from a human cell. Where antigens of human origin areused, they may be obtained from a human tumor cell line, or may beprepared by obtaining a biological sample from a particular patient inquestion. Indeed, methods for the development of antibodies that are"custom-tailored" to the patient's tumor are known (Stevenson et al.,1990) and are contemplated for use in connection with this invention.

(b) Further Tumor Cell Targets and Binding Ligands

In addition to the use of antibodies, other ligands could be employed todirect a coagulating agent to a tumor site by binding to a tumor cellantigen. For tumor antigens that are over-expressed receptors (estrogenreceptor, EGF receptor), or mutant receptors, the corresponding ligandscould be used as targeting agents.

In an analogous manner to endothelial cell receptor ligands, there maybe components that are specifically, or preferentially, bound to tumorcells. For example, if a tumor antigen is an over-expressed receptor,the tumor cell may be coated with a specific ligand in vivo. It seemsthat the ligand could then be targeted either with an antibody againstthe ligand, or with a form of the receptor itself Specific examples ofthese type of targeting agents are antibodies against TIE-1 or TIE-2ligands, antibodies against platelet factor 4, and leukocyte adhesionbinding protein.

ii. Other Disease Targets

In further embodiments, TFs in combination with ITs or coaguligands thatbind to a target molecule that is specifically or preferentiallyexpressed in a disease site other than a tumor site may be employed.

Exemplary target molecules associated with other diseased cells include,for example, leukocyte adhesion molecules, that are associated withpsoriasis; FGF, that is associated with proliferative diabeticretinopathy; platelet factor 4, that is associated with the activatedendothelium of various diseases; and VEGF, that is associated withvascular proliferative disease. It is believed that an animal or patienthaving any one of the above diseases would benefit from the specificinduction of coagulation in the disease site and optionally fromtargeted toxin delivery.

Diseases that are known to have a common angio-dependent pathology, asdescribed in Klagsburn and Folkman (1990), may also be treated asdescribed herein. In particular, a vascular endothelial cell-targetedligand or a stroma-targeted ligand will be used to achieve coagulationin the disease site. The treatment of BPH, diabetic retinopathy,vascular restenosis, vascular adhesions, AVM, meningioma, hemangioma,neovascular glaucoma, rheumatoid arthritis and psoriasis areparticularly contemplated at the present time.

iii. Disease-Associated Vasculature Cell Targets

The cells of the vasculature are intended as targets for use in thepresent invention. In these cases, at least one binding region of theimmunotoxin or coaguligand will be capable of binding to an accessiblemarker preferentially expressed by disease-associated vasculatureendothelial cells. The exploitation of the vascular markers is madepossible due to the proximity of the vascular endothelial cells to thedisease area and to the products of the local aberrant physiologicalprocesses. For example, tumor vascular endothelial cells are exposed totumor cells and tumor-derived products that change the phenotypicprofile of the endothelial cells.

Tumor cells are known to elaborate tumor-derived products, such aslymphokines, monokines, colony-stimulating factors, growth factors andangiogenic factors, that act on the nearby vascular endothelial cells(Kandel et al., 1991;Folkman, 1985a, 1985b) and cytokines (Burrows etal., 1991;Ruco et al., 1990;Borden et al., 1990). The tumor productsbind to the endothelial cells and serve to selectively induce expressionof certain molecules. It is these induced molecules that may be targetedusing the tumor endothelium-specific toxin and/or coagulant deliveryprovided by certain aspects of the present invention. Vascularendothelial cells in tumors proliferate at a rate 30-fold greater thanthose in miscellaneous normal tissues (Denekamp et al., 1982),suggesting that proliferation-linked determinants could also serve asmarkers for tumor vascular endothelial cells.

In certain embodiments of the invention, the targeting component of theimmunotoxins or coaguligands will be a component that has a relativelyhigh degree of specificity for tumor vasculature. These targetingcomponents may be defined as components that bind to molecules expressedon tumor endothelium, but that have little or no expression at thesurface of normal endothelial cells. Such specificity may be assessed bythe standard procedures of immunostaining of tissue sections, which areroutine to those of skill in the art. In terms of coaguligands, it isgenerally proposed that the molecules to be targeted using thebispecific ligands or antibodies of this invention will be those thatare expressed on tumor vasculature at a higher level than on normalendothelial cells.

(a) Vascular Endothelial Cell Markers in Disease

Molecules that are known to be preferentially expressed at the surfaceof vascular endothelial cells in a disease site or environment areherein termed "natural disease-associated vascular endothelial cellmarkers". This term is used for simplicity to refer to the endothelialcell components that are expressed in diseases connected with increasedor inappropriate angiogenesis or endothelial cell proliferation. Oneparticular example are the tumor endothelial cell components that areexpressed in situ in response to tumor-derived factors. These componentsare also termed "naturally-induced tumor endothelial cell markers".

Both VEGF/VPF (vascular endothelial growth factor/vascular permeabilityfactor) and components of the FGF (fibroblast growth factor) family areconcentrated in or on tumor vasculature. The corresponding receptorstherefore provide a potential target for attack on tumor vasculature.For example, VEGF receptors are known to be upregulated on tumorendothelial cells, as opposed to endothelial cells in normal tissues,both in rodents and man (Thieme et al., 1995). Possibly, this is aconsequence of hypoxia--a characteristic of the tumor microenvironment(Leith et al., 1992). FGF receptors are also upregulated three-fold onendothelial cells exposed to hypoxia, and so are believed to beupregulated in tumors (Bicknell and Harris et al., 1992).

The TGF β (transforming growth factor β) receptor (endoglin) onendothelial cells is upregulated on dividing cells, providing anothertarget. One of the present inventors found that endoglin is upregulatedon activated and dividing HUVEC in culture, and is strongly expressed inhuman tissues on endothelial cells at sites of neovascularization,including a broad range of solid tumors and fetal placenta. In contrast,endothelial cells in the majority of miscellaneous non-malignant adulttissues, including preneoplastic lesions, contain little or no endoglin.Importantly, endoglin expression is believed to correlate withneoplastic progression in the breast, as shown by benign fibroadenomasand early carcinomas binding low levels of TEC-4 and TEC-11 antibodies,and late stage intraductal carcinomas and invasive carcinomas bindinghigh levels of these antibodies.

Other natural disease-associated vascular endothelial cell markersinclude a TIE, VCAM-1, P-selectin, E-selectin (ELAM-l), α_(v) β₃integrin, pleiotropin and endosialin, each of which may be targetedusing the invention.

(b) Cytokine-Inducible Vascular Endothelial Markers

Due to the nature of disease processes, which often result in localizeddysfunction within the body, methods are available to manipulate thedisease site whilst leaving other tissues relatively unaffected. This isparticularly true in malignant and benign tumors, which exist asdistinct entities within the body of an animal. For example, the tumorenvironment may be manipulated to create additional markers that arespecific for tumor vascular endothelial cells. These methods generallymimic those that occur naturally in solid tumors, and also involve thelocal production of signaling agents, such as growth factors orcytokines, that induce the specific expression of certain molecules atthe surface of the nearby vascular endothelial cells.

The group of molecules that may be artificially induced to be expressedat the surface of vascular endothelial cells in a disease or tumorenvironment are herein termed "inducible endothelial cell markers", orspecifically, "inducible tumor endothelial cell markers". This term isused to refer to those markers that are artificially induced, i.e.,induced as a result of manipulation by the hand of man, rather thanthose that are induced as part of the disease or tumor developmentprocess in an animal. The term "inducible marker", as defined above, ischosen for simple reference in the context of the present application,notwithstanding the fact that "natural markers" are also induced, e.g.,by tumor-derived agents.

Thus, although not required to practice the invention, techniques forthe selective elicitation of vascular endothelial antigen targets on thesurface of disease-associated vasculature are available that may, ifdesired, be used in conjunction with the invention. These techniquesinvolve manipulating the antigenic expression, or cell surfacepresentation, such that a target antigen is expressed or renderedavailable on the surface of disease-associated vasculature and notexpressed or otherwise rendered accessible or available for binding, orat least to a lesser extent, on the surface of normal endothelium.

Tumor endothelial markers can be induced by tumor-derived cytokines(Burrows et al., 1991;Ruco et al., 1990) and by angiogenic factors(Mignatti et al., 1991). Examples of cell surface markers that may bespecifically induced in the tumor endothelium and then targeted using abispecific coagulating ligand, as provided by the invention, includethose listed in Table III (Bevilacqua et al., 1987;Dustin et al.,1986;Osborn et al., 1989;Collins et al., 1984).

The mechanisms for the induction of the proposed markers; the inducing,or "intermediate cytokine", such as IL-1 and IFN-γ; and the leukocytecell type and associated cytokine-activating molecule, whose targetingwill result in the release of the cytokine, are also set forth in TableIII. In the induction of a specific marker, a bispecific"cytokine-inducing" or "antigen-inducing" antibody is generallyrequired. This antibody will selectively induce the release of theappropriate cytokine in the locale of the tumor, thus selectivelyinducing the expression of the desired target antigen by the vascularendothelial cells. The bispecific antibody cross-links cells of thetumor mass and cytokine-producing leukocytes, thereby activating theleukocytes to release the cytokine.

The preparation and use of bispecific antibodies such as these ispredicated in part on the fact that cross-linking antibodies recognizingCD3, CD14, CD16 and CD28 have previously been shown to elicit cytokineproduction selectively upon cross-linking with the second antigen (Qianet al., 1991). In the context of the present invention, since onlysuccessfully tumor cell-crosslinked leukocytes will be activated torelease the cytokine, cytokine release will be restricted to the localeof the tumor. Thus, expression of the desired marker, such asE-selectin, will be similarly limited to the endothelium of the tumorvasculature.

                                      TABLE III                                   __________________________________________________________________________    POSSIBLE INDUCIBLE VASCULAR TARGETS                                                                                        LEUKOCYTE MOLECULES WHICH,       INDUCIBLE                                    WHEN CROSSLINKED BY              ENDOTHELIAL     SUBTYPES/ALIASES   LEUKOCYTES                                                                              MONOCLONAL ANTIBODIES            CELL      ACRO- (MOLECULAR INDUCING                                                                              WHICH PRODUCE                                                                           ACTIVATE THE CELLS TO            MOLECULES NYM   FAMILY)    CYTOKINES                                                                             THOSE CYTOKINES                                                                         PRODUCE CYTOKINES                __________________________________________________________________________    Endothelial-                                                                            ELAM-1                                                                              --         IL-1, TNF-α,                                                                    monocytes CD14                             Leukocyte       (Selectin) (TNF-β)                                       Adhesion Molecule-1                                                                     E-selectin       (Bacterial                                                                    Endotoxin)                                                                            macrophages                                                                             CD14                                                                mast cells                                                                              FcR for IgE                      Vascular Cell                                                                           VCAM-1                                                                              Inducible Cell Adhesion                                                                  (Bacterial                                                                            monocytes CD14                             Adhesion Molecule-1                                                                           Molecule-110 (INCAM-                                                                     Endotoxin) IL-                                                     110)       1, TNF-α                                                     (Immunoglobulin                                                               Family)                                                                                          macrophages                                                                             CD14                                                                mast cells                                                                              FcR for IgE                                                 TNF-β, IL-4                                                                      helper T cells                                                                          CD2, CD3, CD28                                              TNF     NK cells  FcR for IgG (CD16)               Intercellular                                                                           ICAM-1                                                                              --         IL-1, TNFα                                                                      monocytes CD14                             Adhesion Molecule-1                                                                           (Immunoglobulin                                                                          (Bacterial                                                         Family)    Endotoxin)                                                                            macrophages                                                                             CD15                                                                mast cells                                                                              FcR for IgE                                                 TNF-β, IFNγ                                                                T helper cells                                                                          CD2, CD3, CD28                                                      NK cells  FcR for IgG (CD16)               The Agent for                                                                           LAM-1 MEL-14 Agent (Mouse)                                                                     It-1, TNFα                                                                      monocytes CD14                             Leukocyte Adhesion                                                                      Agent            (Bacterial                                         Molecule-1                 Endotoxin)                                                                            macrophages                                                                             CD14                                                                mast cells                                                                              FcR for IgE                      Major     MHC Class,                                                                          HLA-DR                                                        Human                      IFN-γ                                                                           helper T cells                                                                          CD2, CD3, CD28                   Histocompatability                                                                      II    HLA-DP                                                        Complex Class II                                                                              HLA-DQ                                                        Antigen         I-A                                                           Mouse                                                                                         I-E                                                                                              NK cells  FcR for IgG                      __________________________________________________________________________                                                 (CD16)                       

It is important to note that, from the possible inducible markers listedin Table III, E-selectin and MHC Class II antigens, such as HLA-DR,HLA-DP and HLA-DQ (Collins et al., 1984), are by far the most preferredtargets for use in connection with clinical embodiments. The otheradhesion molecules of Table III appear to be expressed to varyingdegrees in normal tissues, generally in lymphoid organs and onendothelium, making their targeting perhaps appropriate only in animalmodels or in cases where their expression on normal tissues can beinhibited without significant side-effects. The targeting of E-selectinor an MHC Class II antigen is preferred as the expression of theseantigens will likely be the most direct to promote selectively intumor-associated endothelium.

E-selectin

The targeting of an antigen that is not expressed on the surfaces ofnormal endothelium is the most straightforward form of the inductionmethods. E-selectin is an adhesion molecule that is not expressed innormal endothelial vasculature or other human cell types (Cotran et al.,1986), but can be induced on the surface of endothelial cells throughthe action of cytokines such as IL-1, TNF, lymphotoxin and bacterialendotoxin (Bevilacqua et al., 1987). It is not induced by IFN-γ (Wu etal., 1990). The expression of E-selectin may thus be selectively inducedin tumor endothelium through the selective delivery of such a cytokine,or via the use of a composition that causes the selective release ofsuch cytokines in the tumor environment.

Bispecific antibodies are one example of a composition capable ofcausing the selective release of one or more of the foregoing or otherappropriate cytokines in the tumor site, but not elsewhere in the body.Such bispecific antibodies are herein termed "antigen-inducingantibodies" and are, of course, distinct from any bispecific antibodiesof the invention that have targeting and coagulating components.Antigen-inducing antibodies are designed to cross-link cytokine effectorcells, such as cells of monocyte/macrophage lineage, T cells and/or NKcells or mast cells, with tumor cells of the targeted solid tumor mass.This cross-linking would then effect a release of cytokine that islocalized to the site of cross-linking, i.e., the tumor.

Effective antigen-inducing antibodies recognize a selected tumor cellsurface antigen on the one hand and, on the other hand, recognize aselected "cytokine activating" antigen on the surface of a selectedleukocyte cell type. The term "cytokine activating" antigen is used torefer to any one of the various known molecules on the surfaces ofleukocytes that, when bound by an effector molecule, such as an antibodyor a fragment thereof or a naturally-occurring agent or synthetic analogthereof, be it a soluble factor or membrane-bound counter-receptor onanother cell, promotes the release of a cytokine by the leukocyte cell.Examples of cytokine activating molecules include CD14 (the LPSreceptor) and FcR for IgE, which will activate the release of IL-1 andTNFα; and CD16, CD2 or CD3 or CD28, which will activate the release ofIFNγ and TNFβ, respectively.

Once introduced into the bloodstream of an animal bearing a tumor, suchan antigen-inducing bispecific antibody will bind to tumor cells withinthe tumor, cross-link those tumor cells with effector cells, e.g.,monocytes/macrophages, that have infiltrated the tumor, and thereaftereffect the selective release of cytokine within the tumor. Importantly,however, without cross-linking of the tumor and leukocyte, theantigen-inducing antibody will not effect the release of cytokine. Thus,no cytokine release will occur in parts of the body removed from thetumor and, hence, expression of cytokine-induced molecules, e.g.,E-selectin, will occur only within the tumor endothelium.

A number of useful "cytokine activating" antigens are known, which, whencross-linked with an appropriate bispecific antibody, will result in therelease of cytokines by the cross-linked leukocyte. The generallypreferred target for this purpose is CD14, which is found on the surfaceof monocytes and macrophages. When CD14 is cross linked it stimulatesmonocytes/macrophages to release IL-1 (Schutt et al., 1988;Chen et al.,1990), and possibly other cytokines, which, in turn stimulate theappearance of E-selectin on nearby vasculature. Other possible targetsfor cross-linking in connection with E-selectin induction and targetinginclude FcR for IgE, found on Mast cells; FcR for IgG (CD 16), found onNK cells; as well as CD2, CD3 or CD28, found on the surfaces of T cells.Of these, CD14 targeting is generally preferred due to the relativeprevalence of monocyte/macrophage infiltration of solid tumors asopposed to the other leukocyte cell types.

In an exemplary induction embodiment, an animal bearing a solid tumor isinjected with bispecific (Fab'--Fab') anti-CD14/anti-tumor antibody(such as anti-CEA, 9.2.27 antibody against high Mr melanoma antigensOV-TL3 or MOv 18 antibodies against ovarian associated antigens). Theantibody localizes in the tumor, by virtue of its tumor bindingactivity, and then activates monocytes and macrophages in the tumor bycrosslinking their CD14 antigens (Schutt et. al., 1988;Chen et. al.,1990). The activated monocytes/macrophages have tumoricidal activity(Palleroni et. al., 1991) and release IL-1 and TNF which rapidly induceE-selectin antigens on the tumor vascular endothelial cells (Bevilacquaet. al., 1987;Pober et. al., 1991).

MHC Class II Antigens

The second preferred group of inducible markers contemplated for usewith the present invention are the MHC Class II antigens (Collins etal., 1984), including HLA-DR, HLA-DP and HLA-DQ. Class II antigens areexpressed on vascular endothelial cells in most normal tissues inseveral species, including man. Studies in vitro (Collins et al.,1984;Daar et al., 1984; O'Connell et al., 1990) and in vivo (Groenewegenet al., 1985) have shown that the expression of Class II antigens byvascular endothelial cells requires the continuous presence of IFN-γwhich is elaborated by T_(H1) cells and, to a lesser extent, by NK cellsand CD8⁺ T cells.

MHC Class II antigens are not unique to vascular endothelial cells, andare also expressed constitutively on B cells, activated T cells, cellsof monocyte/macrophage lineage and on certain epithelial cells, both inmice (Hammerling, 1976) and in man (Daar et al., 1984). Due to theexpression of MHC Class II antigens on "normal" endothelium, theirtargeting is not quite so straightforward as E-selectin. However, theinduction and targeting of MHC Class II antigens is made possible byusing in conjunction with an immunosuppressant, such as Cyclosporin A(CsA), that has the ability to effectively inhibit the expression ofClass II molecules in normal tissues (Groenewegen et al., 1985). The CsAacts by preventing the activation of T cells and NK cells (Groenewegenet al., 1985;DeFranco, 1991), thereby reducing the basal levels of IFN-γbelow those needed to maintain Class II expression on endothelium.

There are various other cyclosporins related to CsA, includingcyclosporins A, B, C, D, G, and the like, that also haveimmunosuppressive action and are likely to demonstrate an ability tosuppress Class II expression. Other agents that might be similarlyuseful include FK506 and rapamycin.

Thus, the practice of the MHC Class II induction and targetingembodiment requires a pretreatment of the tumor-bearing animal with adose of CsA or other Class II immunosuppressive agent that is effectiveto suppress Class II expression. In the case of CsA, this will typicallybe on the order of about 10 to about 30 mg/kg body weight. Oncesuppressed in normal tissues, Class II antigens can then be selectivelyinduced in the tumor endothelium, again through the use of a bispecificantibody.

In this case, the antigen-inducing bispecific antibody will havespecificity for a tumor cell marker and for an activating antigen foundon the surface of an effector cell that is capable of inducing IFN-γproduction. Such effector cells will generally be helper T cells (T_(H))or Natural Killer (NK) cells. In these embodiments, it is necessary thatT cells, or NK cells if CD16 is used, be present in the tumor to producethe cytokine intermediate in that Class II antigen expression isachieved using IFN-γ, but is not achieved with the other cytokines.Thus, for the practice of this aspect of the invention, one will desireto select CD2, CD3, CD28, or most preferably CD28, as the cytokineactivating antigen for targeting by the antigen-inducing bispecificantibody.

The T cells that should be activated in the tumor are those adjacent tothe vasculature since this is the region most accessible to cells and isalso where the bispecific antibody will be most concentrated. Theactivated T cells should then secrete IFN-γ which induces Class IIantigens on the adjacent tumor vasculature.

The use of a bispecific (Fab'--Fab') antibody having one arm directedagainst a tumor antigen and the other arm directed against CD28 iscurrently preferred. This antibody will cross-link CD28 antigens on Tcells in the tumor which, when combined with a second signal (provided,for example, by IL-1 which is commonly secreted by tumor cells (Burrowset al., 1991;Ruco et al, 1990), has been shown to activate T cellsthrough a CA²⁺ -independent non-CsA-inhibitable pathway (Hess et al.,1991;June et al., 1987;Bjorndahl et al., 1989).

The preparation of antibodies against various cytokine activatingmolecules is also well known in the art. For example, the preparationand use of anti-CD14 and anti-CD28 monoclonal antibodies having theability to induce cytokine production by leukocytes has now beendescribed by several laboratories (reviewed in Schutt et al., 1988;Chenet al., 1990, and June et al., 1990, respectively). Moreover, thepreparation of monoclonal antibodies that will stimulate leukocyterelease of cytokines through other mechanisms and other activatingantigens is also known (Clark et al., 1986;Geppert et al., 1990).

In still further embodiments, the inventors contemplate an alternativeapproach for suppressing the expression of Class II molecules, andselectively eliciting Class II molecule expression in the locale of thetumor. This approach, which avoids the use of both CsA and a bispecificactivating antibody, takes advantage of the fact that the expression ofClass II molecules can be effectively inhibited by suppressing IFN-γproduction by T cells, e.g., through use of an anti-CD4 antibody (Streetet al., 1989). Using this embodiment, IFN-γ production is inhibited byadministering anti-CD4, resulting in the general suppression of Class IIexpression. Class II is then induced only in the tumor site, e.g., usingtumor-specific T cells which are only activatable within the tumor.

In this mode of treatment, one will generally pretreat an animal orhuman patient with a dose of anti-CD4 that is effective to suppressIFN-γ production and thereby suppress the expression of Class IImolecules. Effective doses are contemplated to be, for example, on theorder of about 4 to about 10 mg/kg body weight. After Class IIexpression is suppressed, one will then prepare and introduce into thebloodstream an IFN-γ-producing T cell clone (e.g., T_(h) 1 or cytotoxicT lymphocyte, CTL) specific for an antigen expressed on the surface ofthe tumor cells. These T cells localizes to the tumor mass, due to theirantigen recognition capability and, upon such recognition, then releaseIFN-γ. In this manner, cytokine release is again restricted to thetumor, thus limiting the expression of Class II molecules to the tumorvasculature.

The IFN-γ-producing T cell clone may be obtained from the peripheralblood (Mazzocchi et al., 1990), however, a preferred source is fromwithin the tumor mass (Fox et al., 1990). The currently preferred meansof preparing such a T cell clone is to remove a portion of the tumormass from a patient; isolate cells, using collagenase digestion, wherenecessary; enrich for tumor infiltrating leukocytes using densitygradient centrifugation, followed by depletion of other leukocytesubsets by, e.g., treatment with specific antibodies and complement; andthen expand the tumor infiltrating leukocytes in vitro to provide theIFN-γ producing clone. This clone will necessarily be immunologicallycompatible with the patient, and therefore should be well tolerated bythe patient.

It is proposed that particular benefits will be achieved by furtherselecting a high IFN-γ producing T cell clone from the expandedleukocytes by determining the cytokine secretion pattern of eachindividual clone every 14 days. To this end, rested clones will bemitogenically or antigenically-stimulated for about 24 hours and theirculture supernatants assayed, e.g., using a specific sandwich ELISAtechnique (Cherwinski et al., 1989), for the presence of IL-2, IFN-γ,IL-4, IL-5 and IL-10. Those clones secreting high levels of IL-2 andIFN-γ, the characteristic cytokine secretion pattern of T_(H1) clones,will be selected. Tumor specificity will be confirmed usingproliferation assays.

Furthermore, one will prefer to employ as the anti-CD4 antibody ananti-CD4 Fab, because it will be eliminated from the body within 24hours after injection and so will not cause suppression of thetumor-recognizing T-cell clones that are subsequently administered. Thepreparation of T cell clones having tumor specificity is generally knownin the art, as exemplified by the production and characterization of Tcell clones from lymphocytes infiltrating solid melanoma tumors (Maedaet al., 1991).

In using either of the MHC Class II suppression-induction methods,additional benefits will likely result from the fact that anti-Class IIantibodies injected intravenously do not appear to reach the epithelialcells or the monocytes/macrophages in normal organs other than the liverand spleen. Presumably this is because the vascular endothelium in mostnormal organs is tight, not fenestrated as it is in the liver andspleen, and so the antibodies must diffuse across basement membranes toreach the Class II-positive cells. Also, any B cell elimination that mayresult, e.g., following cross-linking, is unlikely to pose a significantproblem as these cells are replenished from Class II negativeprogenitors (Lowe et al., 1986). Even B cell killing, as occurs in Blymphoma patients, causes no obvious harm (Vitetta et al., 1991).

In summary, although the tumor coagulating compositions and antibodiesof the present invention are elegantly simple, and do not require theinduction of antigens for their operability, the combined use of anantigen-inducing bispecific antibody with this invention is alsocontemplated. Such antibodies would generally be administered prior tothe bispecific coagulating ligands of this invention.

Generally speaking, the more "immunogenic" tumors would be more suitablefor the MHC Class II approach involving, e.g., the cross-linking of Tcells in the tumor through an anti-CD28/anti-tumor bispecific antibody,because these tumors are more likely to be infiltrated by T cells, aprerequisite for this method to be effective. Examples of immunogenicsolid tumors include renal carcinomas, melanomas, a minority of breastand colon cancers, as well as possibly pancreatic, gastric, liver, lungand glial tumor cancers. These tumors are referred to as "immunogenic"because there is evidence that they elicit immune responses in the hostand they have been found to be amenable to cellular immunotherapy(Yamaue et al., 1990). In the case of melanomas and large bowel cancers,the most preferred antibodies for use in these instances would be B72.3(anti-TAG-72) and PRSC5/PR4C2 (anti-Lewis a) or 9.2.27 (anti-high Mrmelanoma antigen).

For the majority of solid tumors of all origins, an anti-CD14 approachthat employs a macrophage/monocyte intermediate would be more suitable.This is because most tumors are rich in macrophages. Examples ofmacrophage-rich tumors include most breast, colon and lung carcinomas.Examples of preferred anti-tumor antibodies for use in these instanceswould be anti-HER-2, B72.3, SM-3, HMFG-2, and SWA 11 (Smith et al.,1989).

(c) Coagulant-Inducible Markers

Coagulants, such as thrombin, Factor IX/IXa, Factor X/Xa, plasmin andmetalloproteinases, such as interstitial collagenases, stromelysins andgelatinases, also act to induce certain markers. In particular,E-selectin, P-selectin, PDGF and ICAM-1 are induced by thrombin (Sugamaet. al., 1992;Shankar et. al., 1994).

Therefore, for this induction, an anti-coagulant/anti-tumor bispecificantibody will be utilized. The antibody will localize in the tumor viaits tumor binding activity. The bispecific will then concentrate thecoagulant, e.g., thrombin, in the tumor, resulting in induction ofE-selectin and P-selectin on the tumor vascular endothelial cells(Sugama et. al., 1991;Shankar et. al., 1994).

Alternatively, targeting of truncated Tissue Factor to tumor cells orendothelium will induce thrombin deposition within the tumor. As thethrombin is deposited, E-selectin and P-selectin will be induced on thetumor vascular endothelial cells.

(d) Antibodies to Vascular Endothelial Cell Markers

A straightforward means of recognizing a disease-associated vasculaturetarget, whether induced in the natural environment or by artificialmeans, is through the use of an antibody that has binding affinity forthe particular cell surface receptor, molecule or antigen. These includeantibodies directed against all cell surface components that are knownto be present on, e.g., tumor vascular endothelial cells, those that areinduced or over-expressed in response to tumor-derived factors, andthose that are induced following manipulation by the hand of man.

Anti-vWF recognizes the antigen VIII R Ag and stains 100% of tumor typespresented and stains 100% of the vessels in the tumor and presents astrong staining pattern in normal vessels. FB5 recognizes the antigenendosialin and stains 50% of tumor types presented and stains 10-30% ofthe vessels in the tumor and presents a staining pattern in normalvessels in the lymphoid organs. TP3 recognizes the antigen 80 kDaosteosarcoma related antigen protein and stains 50% of tumor typespresented and stains 10-30% of the vessels in the tumor and presents astrong staining pattern in normal vessels on the small blood vessels.BC-1 recognizes the antigen fibronectin isoforms and stains 60% of tumortypes presented and stains 10-30% of the vessels in the tumor andpresents no staining pattern in normal vessels. TV-1 recognizes theantigen fibronectin and stains 100% of tumor types presented and stains100% of the vessels in the tumor and presents a strong staining patternin all normal vessels. LM 609 recognizes the α_(v) β_(e) vitronectinreceptor and stains 85% of tumor types presented and stains 70-80% ofthe vessels in the tumor and presents a medium staining pattern innormal vessels. TEC-11 recognizes endoglin and stains 100% of tumortypes presented and stains 100% of the vessels in the tumor and presenta weak staining pattern in most normal vessels. TEC 110 recognizesantigens VEGF and stains 100% of tumor types presented and stains 100%of the vessels in the tumor and present a weak staining pattern in mostnormal vessels.

In a comparative study of anti-EC mAbs on human tumors it was found thatTEC 110, TV-1, and TEC 11, were positive in gastrointestinal, parotid,breast, ovarian uterine, lung and Hodgkin's type tumors. Whereas FB-5had a slight staining in gastrointestinal and lung tumors and wasnegative in the other tumors listed. TP-3 was positive ingastrointestinal tumors and less so in parotid tumor types, ovarian andHodgkins type tumors. BC-1 was positive for gastrointestinal tumors aswells as the reproductive and respiratory tumors IM 609 was positive ingastrointestinal, ovarian, uterine lung and Hodgkin's tumors as wells asthe reproductive, and respiratory tumors.

Two further antibodies that may be used in this invention are thosedescribed by Rettig et al. (1992) and Wang et al. (1993) that aredirected against unrelated antigens of unknown function expressed in thevasculature of human tumors, but not in most normal tissues.

The antibody described by Kim et. al. (1993) may also be used in thisinvention, particularly as this antibody inhibited angiogenesis andsuppressed tumor growth in vivo.

Antibodies that have not previously been shown to be specific for humantumors may also be used. For example, Venkateswaran et al. (1992)described the production of anti-FGF MAbs. Xu et. al. (1992) developedand characterized a panel of 16 isoform and domain-specific polyclonaland monoclonal antibodies against FGF receptor (flg) isoforms. Massogliaet al. (1987) also reported MAbs against the FGF receptor.

(e) Generation of Antibodies to Disease Vasculature

In addition to utilizing a known antibody, such as those described aboveand others known and published in the scientific literature, one mayalso generate a novel antibody using standard immunization procedures,as described in more detail hereinbelow. To generate an antibody againsta known disease-associated vascular marker antigen, one would immunizean animal with an immunogenic composition comprising the antigen. Thismay be a membrane preparation that includes, or is enriched for, theantigen; a relatively purified form of the antigen, as isolated fromcells or membranes; a highly purified form of the antigen, as obtainedby a variety of purification steps using, e.g., a native antigen extractor a recombinant form of the antigen obtained from a recombinant hostcell.

The present invention also provides yet further methods for generatingan antibody against an antigen present on disease-associated vasculatureendothelial cells, which methods are suitable for use even where thebiochemical identity of the antigen remains unknown. These methods areexemplified through the generation of an antibody against tumorvasculature endothelial cells. A first means of achieving antibodygeneration in this manner uses a preparation of vascular endothelialcells obtained from the tumor site of an animal or human patient. Onesimply immunizes an experimental animal with a preparation of such cellsand collects the antibodies so produced. The most useful form of thismethod is that where specific antibodies are subsequently selected, asmay be achieved using conventional hybridoma technology and screeningagainst tumor vascular endothelial cells.

A development of the above method is that which mimics the tumorvasculature phenomenon in vitro, and where cell purification is notnecessary. In using this method, endothelial cells are subjected totumor-derived products, such as might be obtained from tumor-conditionedmedia, in cell culture rather than in an animal. This method generallyinvolves stimulating endothelial cells with tumor-conditioned medium andemploying the stimulated endothelial cells as immunogens to prepare acollection of antibodies. Again, specific antibodies should be selected,e.g., using conventional monoclonal antibody technology, or othertechniques such as combinatorial immunoglobulin phagemid librariesprepared from RNA isolated from the spleen of the immunized animal. Onewould select a specific antibody that preferentially recognizestumor-stimulated vascular endothelium and reacts more strongly withtumor-associated endothelial cells than with normal adult human tissues.

(f) Anti-Endoglin Antibodies

Antibodies having relative specificity for tumor vascular endotheliumhave been prepared and isolated by one of the inventors. The MAbs termedtumor endothelial cell antibody 4 and tumor endothelial cell antibody 11(TEC4 and TEC11) were obtained using the above method (U.S. Ser. Nos.08/457,229 and 08/457,031, each incorporated herein by reference). Theantigen recognized by TEC4 and TEC11 was ultimately determined to be themolecule endoglin. The epitopes on endoglin recognized by TEC4 and TEC11are present on the cell surface of stimulated HUVE cells, and onlyminimally present (or immunologically accessible) on the surface ofnon-stimulated cells. MAbs have previously been raised against endoglin.However, analyzing the reactivity with HUVEC or TCM-activated HUVEC cellsurface determinants by FACS or indirect immunofluorescence shows theepitopes recognized by TEC-4 and TEC-11 to be distinct from those of aprevious antibody termed 44G4 (Gougos and Letarte, 1988).

(g) Use of Vascular Endothelial Cell Binding Ligands

Biological ligands that are known to bind or interact with endothelialcell surface molecules, such as growth factor receptors, may also beemployed as a targeting component.

The growth factors or ligands contemplated to be useful as targets inthis sense include VEGF/VPF, FGF, TGFβ, ligands that bind to a TIE,tumor-associated fibronectin isoforms, scatter factor, hepatocyte growthfactor (HGF), platelet factor 4 (PF4), PDGF and TIMP.

Particularly preferred targets are VEGF/VPF, the FGF family of proteinsand TGFβ. Abraham et al. (1986) cloned FGF, which is therefore availableas a recombinant protein. As reported by Ferrara et al. (1991), fourspecies of VEGF having 121, 165, 189, and 206 amino acids have beencloned.

(h) Targeting of Bound Ligands

Antibodies or specific targeting ligands may also be directed to anycomponent that binds to the surface of vascular endothelial cells in adisease site, such as a tumor. Such components are exemplified bytumor-derived ligands and antigens, such as growth factors, that bind tospecific cell surface receptors already present on the endothelialcells, or to receptors that have been induced, or over-expressed, onsuch cells in response to the tumor environment. Tumorvasculature-associated targets may also be termed tumor-derivedendothelial cell binding factors.

A level of specificity required for successful disease targeting will beachieved partly because the local endothelial cells will be induced toexpress, or reveal, receptors that are not present, or areunder-expressed or masked, on normal endothelial cells. With tumors,further specificity will result due to the fact that endothelial cellsin the tumor will capture the tumor-derived factors, and bind them tothe cell surface, reducing the amount of ligand available for othertissues. When combined with the further dilution of the factor or ligandby distribution in the blood and tissue fluid pool, endothelial cells innormal tissues will be expected to bind relatively little of suchfactors. Thus, operationally, cell-surface bound ligands or factors willbe able to used as a tumor endothelial cell marker.

In addition to manufacture by the tumor cells themselves, tumorendothelial cell binding factors may also originate from other celltypes, such as macrophages and mast cells, that have infiltrated tumors,or may be elaborated by platelets that become activated within thetumor.

Further growth factors or ligands contemplated to be useful as tumorvasculature-associated targeting agents include EGF, FGF, VEGF, TGFβ,HGF (NaKamura, 1991), angiotropin, TGF-α, TNF-α, PD-ECGF and TIE bindingligands (Bicknell and Harris, 1992). The currently preferred targetingagents are VEGF/VPF, the FGF family of proteins, transforming growthfactor-β (TGF-β); TGF-α; tumor necrosis factor-α (TNF-α); angiotropin;platelet-derived endothelial cell growth factor (PD-ECGF); TIE bindingligands; pleiotropin. In addition, non-antibody targeting components,such as annexins and peptides comprising the tripeptide sequence R-G-D,which specifically target the tumor vasculature (Pasqualini et al.,1997), are also contemplated for use in certain aspects of theinvention.

Another aspect of the present invention is the use of targetingantibodies, or binding regions therefrom, that are specific for epitopespresent only on ligand-receptor complexes, which epitopes are absentfrom both the individual (free) ligand and the receptor in its unboundform. These antibodies recognize and bind to the unique conformationthat results when a ligand, such as a growth factor, binds to itsreceptor, such as a growth factor receptor, to form a specifically boundcomplex. Such epitopes are not present on the uncomplexed forms of theligands or receptors.

The inventors contemplate that the ligand-receptor complexes to whichthese antibodies bind are present in significantly higher number ontumor-associated endothelial cells than on non-tumor associatedendothelial cells. Such antibodies will therefore be useful as targetingagents and will serve to further increase the specificity of thebispecific coagulants of the invention.

(i) Receptor Constructs

Soluble binding domains of endothelial cell surface receptors are alsocontemplated for use as targeting ligands in the present invention. Thisconcept is generally based upon the well-known sandwich bindingphenomena that has been exploited in a variety of in vitro and in vivobinding protocols. Basically, as the endothelial cells express specificreceptors, the cells bind to and adsorb the corresponding ligands, theligands are then available for binding to further receptor constructsshould they be introduced into the system.

A range of useful endothelial cell receptors has been identified in theforegoing sections, with VEGF/VPF, FGF, TGFβ, TIE-1 and TIE-2 beingparticularly preferred targets. Each of these receptors could bemanipulated to form a soluble binding domain for use as a targetingligand.

iv. Disease-Associated Stromal Cell Targets

(a) Extracellular Matrix/Stromal Targets

The usefulness of the basement membrane markers in tumoral pathology wasdescribed by Birembaut et al. (1985). These studies showed that thedistribution of basement membrane (BM) markers, type IV collagen,laminin (LM), heparan sulphate proteoglycan (HSP) and fibronectin (FN)were disrupted in tumoral pathology. Burtin et. al. (1983) alsodescribed alterations of the basement membrane and connective tissueantigens in human metastatic lymph nodes.

A preferred target for use with the invention is RIBS. Ugarova et al.(1993) reported that conformational changes occur in fibrinogen and areelicited by its interaction with the platelet membrane glycoproteinGPIIb-IIIa. The binding of fibrinogen to membrane glycoproteinGPIIb-IIIa on activated platelets leads to platelet aggregation. Thisinteraction results in conformational changes in fibrinogen as evidencedby the expression of receptor-induced binding sites, RIBS, epitopeswhich are expressed by the bound but not the free ligand.

Two RIBS epitopes have been localized by Ugarova et al. (1993). Onesequence resides at γ112-119 and is recognized by MAb 9F9;the second isthe RGDF sequence at Aα 95-98 and is recognized by mAb 155B16. Theseepitopes are also exposed by adsorption of fibrinogen onto a plasticsurface and digestion of the molecule by plasmin. Proteolytic exposureof the epitopes coincides with cleavage of the carboxyl-terminal aspectsof the Aα-chains to form fragment X₂. The inaccessibility of the RGDFsequence at Aα 95-98 in fibrinogen suggests that this sequence does notparticipate in the initial binding of the molecule to GPIIb-IIIa.

Binding of fibrinogen to its receptor alters the conformation of thecarboxyl-terminal aspects of the Aα-chains, exposing the sequences whichreside in the coiled-coil connector segments between the D and E domainsof the molecule, generating the RIBS epitopes. In practical terms, theRIBS sequences are proposed as epitopes for use in targeting with acoaguligand. The MAbs 9F9 and 155B16 may thus be advantageously used, asmay the antibodies described by Zamarron et al. (1991).

(b) Additional Cellular Targets

The combinations for use in the present invention have the furtheradvantage that they may be used to direct coagulants todisease-associated vasculature by targeting them to cell types foundwithin the disease region.

Platelets participate in hemostasis and thrombosis by adhering toinjured blood vessel walls and accumulating at the site of injury.Although platelet deposition at sites of blood vessel injury isresponsible for the primary arrest of bleeding under physiologicconditions, it can lead to vascular occlusion with ensuing ischemictissue damage and thrombus embolization under pathologic conditions.

Interactions of platelets with their environment and with each otherrepresent complex processes that are initiated at the cell surface. Thesurface membrane, therefore, provides a reactive interface between theexternal medium, including components of the blood vessel wall andplasma, and the platelet interior.

p-155, a multimeric platelet protein that is expressed on activatedplatelets (Hayward et al., 1991), may be targeted using the invention.Platelets respond to a large number of stimuli by undergoing complexbiochemical and morphological changes. These changes are involved inphysiological processes including adhesion, aggregation, andcoagulation. Platelet activation produces membrane alterations that canbe recognized by monoclonal antibodies. The monoclonal antibody JS-1(Hayward et al., 1991) is one such antibody contemplated for use as partof a coaguligand.

Ligand-induced binding sites (LIBS) are sites expressed on cell surfacereceptors only after ligand binding causes the receptor to change shape,mediate subsequent biological events. These may be seen as counterpartsto RIBS and are also preferred targets for use with the presentinvention.

13 anti-LIBS antibodies have been developed by Frelinger et. al. (1990;1991), any one of which may be used to deliver a coagulant to a diseaseor tumor site in accordance herewith. The murine monoclonalanti-platelet antibodies MA-TSPI-1 (directed against humanthrombospondin) and MA-PMI-2, MA-PMI-1, and MA-LIBS-1 (directed againstLIBS on human platelet glycoprotein IIb/IIIa) of Dewerchin et al. (1991)may also be used, as may RUU 2.41 and LIBS-1 of Heynen et al. (1994);OP-G2 of Tomiyama et al. (1992); and Ab-15.

Many other targets, such as antigens on smooth muscle cells, pericytes,fibroblasts, macrophages and infiltrating lymphocytes and leukocytes mayalso be used.

v. Toxins

For certain applications, it is envisioned that the second therapeuticagents will be pharmacological agents attached to antibodies or growthfactors, particularly cytotoxic or otherwise anti-cellular agents havingthe ability to kill or suppress the growth or cell division ofendothelial cells. In general, the secondary aspects of the inventioncontemplate the use of any pharmacological agent that can be conjugatedto a targeting agent, preferably an antibody, and delivered in activeform to the targeted endothelium or stroma. Exemplary anti-cellularagents include chemotherapeutic agents, radioisotopes as well ascytotoxins. In the case of chemotherapeutic agents, the inventorspropose that agents such as a hormone such as a steroid; ananti-metabolite such as cytosine arabinoside, fluorouracil, methotrexateor aminopterin; an anthracycline; mitomycin C; a vinca alkaloid;demecolcine; etoposide; mithramycin; or an anti-tumor alkylating agentsuch as chlorambucil or melphalan, will be particularly preferred. Otherembodiments may include agents such as a cytokine, growth factor,bacterial endotoxin or the lipid A moiety of bacterial endotoxin. In anyevent, it is proposed that agents such as these may, if desired, besuccessfully conjugated to a targeting agent, preferably an antibody, ina manner that will allow their targeting, internalization, release orpresentation to blood components at the site of the targeted endothelialcells as required using known conjugation technology (see, e.g., Ghoseet al., 1983 and Ghose et al., 1987).

In certain preferred embodiments, the immunotoxins will includegenerally a plant-, fungus- or bacteria-derived toxin, such as an Achain toxins, a ribosome inactivating protein, α-sarcin, aspergillin,restrictocin, a ribonuclease, diphtheria toxin or pseudomonas exotoxin,to mention just a few examples. The use of toxin-antibody constructs iswell known in the art of immunotoxins, as is their attachment toantibodies. Of these, a particularly preferred toxin for attachment toantibodies will be a deglycosylated ricin A chain. Deglycosylated ricinA chain is preferred because of its extreme potency, longer half-life,and because it is economically feasible to manufacture it a clinicalgrade and scale.

(a) Preparation of Targeting Agent-Toxin Conjugates

While the preparation of immunotoxins is, in general, well known in theart (see, e.g., patents U.S. Pat. No. 4,340,535, and EP 44167, bothincorporated herein by reference), the inventors are aware that certainadvantages may be achieved through the application of certain preferredtechnology, both in the preparation of the immunotoxins and in theirpurification for subsequent clinical administration. For example, whileIgG based immunotoxins will typically exhibit better binding capabilityand slower blood clearance than their Fab' counterparts, Fab'fragment-based immunotoxins will generally exhibit better tissuepenetrating capability as compared to IgG based immunotoxins.

Additionally, while numerous types of disulfide-bond containing linkersare known which can successfully be employed to conjugate the toxinmoiety with the targeting agent, certain linkers will generally bepreferred over other linkers, based on differing pharmacologicalcharacteristics and capabilities. For example, linkers that contain adisulfide bond that is sterically "hindered" are to be preferred, due totheir greater stability in vivo, thus preventing release of the toxinmoiety prior to binding at the site of action. Furthermore, whilecertain advantages in accordance with the invention will be realizedthrough the use of any of a number of toxin moieties, the inventors havefound that the use of ricin A chain, and even more preferablydeglycosylated A chain, will provide particular benefits.

A wide variety of cytotoxic agents are known that may be conjugated toanti-endothelial cell antibodies. Examples include numerous usefulplant-, fungus- or even bacteria-derived toxins, which, by way ofexample, include various A chain toxins, particularly ricin A chain,ribosome inactivating proteins such as saporin or gelonin, α-sarcin,aspergillin, restrictocin, ribonucleases such as placental ribonuclease,angiogenic, diphtheria toxin, and pseudomonas exotoxin, to name just afew. The most preferred toxin moiety for use in connection with theinvention is toxin A chain which has been treated to modify or removecarbohydrate residues, so called deglycosylated A chain. The inventorshave had the best success through the use of deglycosylated ricin Achain (dgA) which is now available commercially from InlandLaboratories, Austin, Tex.

However, it may be desirable from a pharmacological standpoint to employthe smallest molecule possible that nevertheless provides an appropriatebiological response. One may thus desire to employ smaller A chainpeptides which will provide an adequate anti-cellular response. To thisend, it has been discovered by others that ricin A chain may be"truncated" by the removal of 30 N-terminal amino acids by Nagarase(Sigma), and still retain an adequate toxin activity. It is proposedthat where desired, this truncated A chain may be employed in conjugatesin accordance with the invention.

Alternatively, one may find that the application of recombinant DNAtechnology to the toxin A chain moiety will provide additionalsignificant benefits in accordance the invention. In that the cloningand expression of biologically active ricin A chain has now been enabledthrough the publications of others (O'Hare et al., 1987;Lamb et al.,1985;Halling et al., 1985), it is now possible to identify and preparesmaller or otherwise variant peptides which nevertheless exhibit anappropriate toxin activity. Moreover, the fact that ricin A chain hasnow been cloned allows the application of site-directed mutagenesis,through which one can readily prepare and screen for A chain derivedpeptides and obtain additional useful moieties for use in connectionwith the present invention.

The cross-linking of the toxin A chain region of the conjugate with thetargeting agent region is an important aspect of the invention. Incertain cases, it is required that a cross-linker which presentsdisulfide function be utilized for the conjugate to have biologicalactivity. The reason for this is unclear, but is likely due to a needfor certain toxin moieties to be readily releasable from the targetingagent once the agent has "delivered" the toxin to the targeted cells.Each type of cross-linker, as well as how the cross-linking isperformed, will tend to vary the pharmacodynamics of the resultantconjugate. Ultimately, in cases where a releasable toxin iscontemplated, one desires to have a conjugate that will remain intactunder conditions found everywhere in the body except the intended siteof action, at which point it is desirable that the conjugate have good"release" characteristics. Therefore, the particular cross-linkingscheme, including in particular the particular cross-linking reagentused and the structures that are cross-linked, will be of somesignificance.

Depending on the specific toxin compound used as part of the fusionprotein, it may be necessary to provide a peptide spacer operativelyattaching the targeting agent and the toxin compound which is capable offolding into a disulfide-bonded loop structure. Proteolytic cleavagewithin the loop would then yield a heterodimeric polypeptide wherein thetargeting agent and the toxin compound are linked by only a singledisulfide bond. See, for example, Lord et al. (1992). An example of sucha toxin is a Ricin A-chain toxin.

When certain other toxin compounds are utilized, a non-cleavable peptidespacer may be provided to operatively attach the targeting agent and thetoxin compound of the fusion protein. Toxins which may be used inconjunction with non-cleavable peptide spacers are those which may,themselves, be converted by proteolytic cleavage, into a cytotoxicdisulfide-bonded form (see for example, Ogata et al., 1990). An exampleof such a toxin compound is a Pseudonomas exotoxin compound.

Nucleic acids that may be utilized herein comprise nucleic acidsequences that encode a targeting agent of interest and nucleic acidsequences that encode a toxin agent of interest. Such targetagent-encoding and toxin agent-encoding nucleic acid sequences areattached in a manner such that translation of the nucleic acid yieldsthe targeting agent/toxin compounds of the invention.

(b) Attachment of Other Agents to Targeting Agents

It is contemplated that most therapeutic applications of the additionalIT aspects of the present invention will involve the targeting of atoxin moiety to the tumor endothelium or stroma. This is due to the muchgreater ability of most toxins to deliver a cell killing effect ascompared to other potential agents. However, there may be circumstances,such as when the target antigen does not internalize by a routeconsistent with efficient intoxication by targeting agent/toxincompounds, such as immunotoxins, where one will desire to targetchemotherapeutic agents such as anti-tumor drugs, other cytokines,antimetabolites, alkylating agents, hormones, and the like. Theadvantages of these agents over their non-targeting agent conjugatedcounterparts is the added selectivity afforded by the targeting agent,such as an antibody. One might mention by way of example agents such assteroids, cytosine arabinoside, methotrexate, aminopterin,anthracyclines, mitomycin C, vinca alkaloids, demecolcine, etoposide,mithramycin, and the like. This list is, of course, merely exemplary inthat the technology for attaching pharmaceutical agents to targetingagents, such as antibodies, for specific delivery to tissues is wellestablished (see, e.g., Ghose and Blair, 1987).

A variety of chemotherapeutic and other pharmacological agents have nowbeen successfully conjugated to antibodies and shown to functionpharmacologically (see, e.g., Vaickus et al., 1991). Exemplaryantineoplastic agents that have been investigated include doxorubicin,daunomycin, methotrexate, vinblastine, and various others (Dillman etal., 1988; Pietersz et al., 1988). Moreover, the attachment of otheragents such as neocarzinostatin (Kimura et al., 1983), macromycin(Manabe et al., 1984), trenimon (Ghose, 1982) and α-amanitin (Davis andPreston, 1981) has been described.

vi. Coaguligands

The second, targeted agent for optional use with the invention may alsocomprise a targeted component that is capable of promoting coagulation.Such "targeted coagulation promoting agents" or "coaguligands" includeany of the foregoing targeting agents that are operably associated withone or more coagulation factors. The targeting agent may be directlylinked to a factor that directly or indirectly stimulates coagulation,or the targeting agent may linked to a second binding region that iscapable of binding and releasing a coagulation factor that directly orindirectly stimulates coagulation.

(a) Coagulation Factors

Exemplary coagulation factors are the types of tTF, dimeric, multimericand mutant molecules of the present invention, as described in detailherein.

A variety of other coagulation factors may be used in connection withthe present invention, as exemplified by the agents set forth below.Where a coagulation factor is covalently linked to a first binding ortargeting agent, a site distinct from its functional coagulating site isused to join the molecules. Appropriate joining regions distinct fromthe active sites, or functional regions, of the coagulation factors arealso described in each of the following sections.

Clotting Factors

Thrombin, Factor V/Va and derivatives, Factor VIII/VIIIa andderivatives, Factor IX/IXa and derivatives, Factor X/Xa and derivatives,Factor XI/XIa and derivatives, Factor XII/XIIa and derivatives, FactorXIII/XIIIa and derivatives, Factor X activator and Factor V activatormay also be used in the present invention.

Venom Coagulants

Russell's viper venom was shown to contain a coagulant protein byWilliams and Esnouf in 1962. Kisiel (1979) isolated a venom glycoproteinthat activates Factor V; and Di Scipio et al. (1977) showed that aprotease from the venom activates human Factor X. The Factor X activatoris the component contemplated for use in this invention.

Monoclonal antibodies specific for the Factor X activator present inRussell's viper venom have also been produced (e.g., MP1 ofPukrittayakamee et al., 1983), and could be used to deliver the agent toa specific target site within the body.

Prostaglandins and Synthetic Enzymes

Thromboxane A₂ is formed from endoperoxides by the sequential actions ofthe enzymes cyclooxygenase and thromboxane synthetase in plateletmicrosomes. Thromboxane A₂ is readily generated by platelets and is apotent vasoconstrictor, by virtue of its capacity to produce plateletaggregation (Whittle et al., 1981).

Both thromboxane A₂ and active analogues thereof are contemplated foruse in the present invention. A synthetic protocol for generatingthromboxane A₂ is described by Bhagwat et al. (1985). The thromboxane A₂analogues described by Ohuchida et. al. (1981) (especially compound 2)are particularly contemplated for use herewith.

It is possible that thromboxane synthase, and other enzymes thatsynthesize platelet-activating prostaglandins, may also be used as"coagulants" in the present context. Shen and Tai (1986) describemonoclonal antibodies to, and immunoaffinity purification of,thromboxane synthase; and Wang et. al. (1991) report the cDNA for humanthromboxane synthase.

Inhibitors of Fibrinolysis

α2-antiplasmin, or α2-plasmin inhibitor, is a proteinase inhibitornaturally present in human plasma that functions to efficiently inhibitthe lysis of fibrin clots induced by plasminogen activator (Moroi andAoki, 1976). α2-antiplasmin is a particularly potent inhibitor, and iscontemplated for use in the present invention.

α2-antiplasmin may be purified as first described by Moroi and Aoki(1976). Other purification schemes are also available, such as usingaffinity chromatography on plasminogen-Sepharose, ion-exchangechromatography on DEAE-Sephadex and chromatography onConcanavalin-A-Sepharose; or using affinity chromatography on aSepharose column bearing an elastase-digested plasminogen formulationcontaining the three N-terminal triple-loop structures in the plasminA-chain (LBSI), followed by gel filtration (Wiman and Collen,1977;Wiman, 1980, respectively).

As the cDNA sequence for α2-antiplasmin is available (Tone et al.,1977), a preferred method for α2-antiplasmin production will be viarecombinant expression.

Monoclonal antibodies against α2-antiplasmin are also available that maybe used in the bispecific binding ligand embodiments of the invention.For example, Hattey et al. (1987) described two MAbs againstα2-antiplasmin, MPW2AP and MPW3AP. As each of these MAbs were reportedto react equally well with native α2-antiplasmin, they could both beused to deliver exogenous α2-antiplasmin to a target site or to garnerendogenous α2-antiplasmin and concentrate it within the targeted region.Other antibodies, such as JTPI-2, described by Mimuro and colleagues,could also be used.

(b) Agents That Bind Coagulation Factors

Another group of targeted coagulating ligands for use with the TFs ofthis invention are those in which the targeting region is not directlylinked to a coagulation factor, but is linked to a second binding regionthat binds to a coagulating factor.

Where a second binding region is used to bind and deliver a coagulationfactor, the binding region is chosen so that it recognizes a site on thecoagulation factor that does not significantly impair its ability toinduce coagulation. The regions of the coagulation factors suitable forbinding in this manner will generally be the same as those regions thatare suitable for covalent linking to the targeting region, as describedin the previous sections.

However, in that bispecific ligands of this class may be expected torelease the coagulation factor following delivery to the tumor site orregion, there is more flexibility allowed in the regions of thecoagulation factor suitable for binding to a second binding agent orantibody.

Suitable second binding regions for use in this manner, will generallybe antigen combining sites of antibodies that have binding specificityfor the coagulation factor, including functional portions of antibodies,such as scFv, Fv, Fab', Fab and F(ab')₂ fragments.

Bispecific binding ligands that contain antibodies, or fragmentsthereof, directed against Tissuc Factor, Thrombin, Prekallikein, FactorV/Va, Factor VIII/VIIIa, Factor IX/IXa, Factor XIXa, Factor XI/XIa,Factor XII/XIIa, Factor XIII/XIIIa, Russell's viper venom, thromboxaneA₂ or α2-antiplasmin are exemplary embodiments of this aspect of theinvention.

(c) TF Prodrugs

Exemplary tTF prodrugs have the following structures: tTF₁₋₂₁₉ (X)_(n1)(Y)_(n2) Z Ligand, where tTF₁₋₂₁₉ represents TF minus the cytosolic andtransmembrane domains; X represents a hydrophobic transmembrane domainn1 amino acids (AA) in length (n=1-20 AA); Y represents a hydrophilicprotease recognition sequence of n2 AA in length (sufficient AA toensure appropriate protease recognition); Z represents a disulfidethioester or other linking group such as (Cys)₁₋₂ ; Ligand represents anantibody or other targeting moiety recognizing tumor-cells, tumor EC,connective tissue (stroma) or basal lamina markers.

The tTF prodrug is contemplated for injection intravenously allowing itto localize to diseased tissue (e.g., tumor). Once localized in thediseased tissue, endogenous proteases (e.g., metalloproteinases,thrombin, Factor Xa, Factor VIIa, Factor IXa, plasmin) will cleave thehydrophilic protease recognition sequence from the prodrug which willallow the hydrophobic transmembrane sequence to insert into a local cellmembrane. Once the tail has inserted into the membrane, the tTF willregain its coagulation-inducing properties resulting in clot formationin the vasculature of the diseased tissue.

(d) Bispecific Antibodies

In general, the preparation of bispecific antibodies is also well knownin the art, as exemplified by Glennie et al. (1987). Bispecificantibodies have been employed clinically, for example, to treat cancerpatients (Bauer et al., 1991). One method for the preparation ofbispecific antibodies involves the separate preparation of antibodieshaving specificity for the targeted tumor cell antigen, on the one hand,and the coagulating agent (or other desired target, such as anactivating antigen) on the other.

Bispecific antibodies have also been developed particularly for use asimmunotherapeutic agents. As mentioned earlier in conjunction withantigen-induction, certain of these antibodies were developed tocross-link lymphocytes and tumor antigens (Nelson, 1991;Segal et al.,1992). Examples include chimeric molecules that bind T cells, e.g., atCD3, and tumor antigens, and trigger lymphocyte-activation by physicallycross-linking the TCR/CD3 complex in close proximity to the target cell(Staerz et al., 1985;Perez et al., 1985; 1986a;1986b; Ting et al.,1988).

Indeed, tumor cells of carcinomas, lymphomas, leukemias and melanomashave been reported to be susceptible to bispecific antibody-mediatedkilling by T cells (Nelson, 1991;Segal et al., 1992;deLeij et al.,1991). These type of bispecific antibodies have also been used inseveral Phase I clinical trials against diverse tumor targets. Thebispecific cross-linking antibodies may be administered as described inreferences such as deLeij et al. (1991); Clark et al. (1991); Rivoltiniet al. (1992); Bolhuis et al. (1992); and Nitta et al. (1990).

While numerous methods are known in the art for the preparation ofbispecific antibodies, the Glennie et al. (1987) method involves thepreparation of peptic F(ab'γ)₂ fragments from the two chosen antibodies,followed by reduction of each to provide separate Fab'γsh fragments. TheSH groups on one of the two partners to be coupled are then alkylatedwith a cross-linking reagent such as o-phenylenedimaleimide to providefree maleimide groups on one partner. This partner may then beconjugated to the other by means of a thioether linkage, to give thedesired F(ab'γ)₂ heteroconjugate.

Due to ease of preparation, high yield and reproducibility, the Glennieet al. (1987) method is often preferred for the preparation ofbispecific antibodies, however, there are numerous other approaches thatcan be employed and that are envisioned by the inventors. For example,other techniques are known wherein cross-linking with SPDP or protein Ais carried out, or a trispecific construct is prepared (Titus et al.,1987;Tutt et al., 1991).

Another method for producing bispecific antibodies is by the fusion oftwo hybridomas to form a quadroma (Flavell et al., 1991, 1992;Pimm etal., 1992;French et al., 1991;Embleton et al., 1991). As used herein,the term "quadroma" is used to describe the productive fusion of two Bcell hybridomas. Using now standard techniques, two antibody producinghybridomas are fused to give daughter cells, and those cells that havemaintained the expression of both sets of clonotype immunoglobulin genesare then selected.

A preferred method of generating a quadroma involves the selection of anenzyme deficient mutant of at least one of the parental hybridomas. Thisfirst mutant hybridoma cell line is then fused to cells of a secondhybridoma that had been lethally exposed, e.g., to iodoacetamide,precluding its continued survival. Cell fusion allows for the rescue ofthe first hybridoma by acquiring the gene for its enzyme deficiency fromthe lethally treated hybridoma, and the rescue of the second hybridomathrough fusion to the first hybridoma. Preferred, but not required, isthe fusion of immunoglobulins of the same isotype, but of a differentsubclass. A mixed subclass antibody permits the use if an alternativeassay for the isolation of a preferred quadroma.

In more detail, one method of quadroma development and screeninginvolves obtaining a hybridoma line that secretes the first chosen MAband making this deficient for the essential metabolic enzyme,hypoxanthine-guanine phosphoribosyltransferase (HGPRT). To obtaindeficient mutants of the hybridoma, cells are grown in the presence ofincreasing concentrations of 8-azaguanine (1×10⁻⁷ M to 1×10⁻⁵ M). Themutants are subdloned by limiting dilution and tested for theirhypoxanthine/aminopterin/thymidine (HAT) sensitivity. The culture mediummay consist of, for example, DMEM supplemented with 10% FCS, 2 mML-Glutamine and 1 mM penicillin-streptomycin.

A complementary hybridoma cell line that produces the second desired MAbis used to generate the quadromas by standard cell fusion techniques(Galfre et al., 1981), or by using the protocol described by Clark etal. (1988). Briefly, 4.5×10⁷ HAT-sensitive first cells are mixed with2.8×10⁷ HAT-resistant second cells that have been pre-treated with alethal dose of the irreversible biochemical inhibitor iodoacetamide (5mM in phosphate buffered saline) for 30 minutes on ice before fusion.Cell fusion is induced using polyethylene glycol (PEG) and the cells areplated out in 96 well microculture plates. Quadromas are selected usingHAT-containing medium. Bispecific antibody-containing cultures areidentified using, for example, a solid phase isotype-specific ELISA andisotype-specific immunofluorescence staining.

In one identification embodiment to identify the bispecific antibody,the wells of microtiter plates (Falcon, Becton Dickinson Labware) arecoated with a reagent that specifically interacts with one of the parenthybridoma antibodies and that lacks cross-reactivity with bothantibodies. The plates are washed, blocked, and the supernatants (SNs)to be tested are added to each well. Plates are incubated at roomtemperature for 2 hours, the supernatants discarded, the plates washed,and diluted alkaline phosphatase-anti-antibody conjugate added for 2hours at room temperature. The plates are washed and a phosphatasesubstrate, e.g., P-Nitrophenyl phosphate (Sigma, St. Louis) is added toeach well. Plates are incubated, 3N NaOH is added to each well to stopthe reaction, and the OD₄₁₀ values determined using an ELISA reader.

In another identification embodiment, microtiter plates pre-treated withpoly-L-lysine are used to bind one of the target cells to each well, thecells are then fixed, e.g. using 1% glutaraldehyde, and the bispecificantibodies are tested for their ability to bind to the intact cell. Inaddition, FACS, immunofluorescence staining, idiotype specificantibodies, antigen binding competition assays, and other methods commonin the art of antibody characterization may be used in conjunction withthe present invention to identify preferred quadromas.

Following the isolation of the quadroma, the bispecific antibodies arepurified away from other cell products. This may be accomplished by avariety of protein isolation procedures, known to those skilled in theart of immunoglobulin purification. Means for preparing andcharacterizing antibodies are well known in the art (See, e.g.,Antibodies: A Laboratory Manual, 1988).

For example, supernatants from selected quadromas are passed overprotein A or protein G sepharose columns to bind IgG (depending on theisotype). The bound antibodies are then eluted with, e.g. a pH 5.0citrate buffer. The elute fractions containing the BsAbs, are dialyzedagainst an isotonic buffer. Alternatively, the eluate is also passedover an anti-immunoglobulin-sepharose column. The BsAb is then elutedwith 3.5 M magnesium chloride. BsAbs purified in this way are thentested for binding activity by, e.g., an isotype-specific ELISA andimmunofluorescence staining assay of the target cells, as describedabove.

Purified BsAbs and parental antibodies may also be characterized andisolated by SDS-PAGE electrophoresis, followed by staining with silveror Coomassie. This is possible when one of the parental antibodies has ahigher molecular weight than the other, wherein the band of the BsAbsmigrates midway between that of the two parental antibodies. Reductionof the samples verifies the presence of heavy chains with two differentapparent molecular weights.

Furthermore, recombinant technology is now available for the preparationof antibodies in general, allowing the preparation of recombinantantibody genes encoding an antibody having the desired dual specificity(Van Duk et al., 1989). Thus, after selecting the monoclonal antibodieshaving the most preferred binding characteristics, the respective genesfor these antibodies can be isolated, e.g., by immunological screeningof a phage expression library (Oi and Morrison, 1986;Winter andMilstein, 1991). Then, through rearrangement of Fab coding domains, theappropriate chimeric construct can be readily obtained.

vii. Combined Treatment

The Tissue Factor compositions in combination with either immunotoxinsor coaguligands are contemplated for use in the clinical treatment ofvarious human cancers and even other disorders, such as benign prostatichyperplasia and rheumatoid arthritis, in which the intermediate orlonger term arrest of blood flow would be advantageous.

The combination of the Tissue Factor compositions disclosed in thepresent application with immunotoxins and coaguligands are considered tobe particularly useful tools in anti-tumor therapy. From the datapresented herein, including the animal studies, and the knowledge in theart regarding treatment of Lymphoma (Glennie et al., 1988), T-Celltargeting (Nolan and Kennedy, 1990) and drug targeting (Paulus, 1985)appropriate doses and treatment regimens may be straightforwardlydeveloped.

It is currently proposed that effective doses of the immunotoxins andcoaguligands for use with the Tissue Factor constructs described abovein the treatment of cancer will be between about 0.1 mg/kg and about 2mg/kg, and preferably, of between about 0.8 mg/kg and about 1.2 mg/kg,when administered via the IV route at a frequency of about 1 time perweek. Some variation in dosage will necessarily occur depending on thecondition of the subject being treated. The person responsible foradministration will, in any event, determine the appropriate dose forthe individual subject. Such optimization and adjustment is routinelycarried out in the art and by no means reflects an undue amount ofexperimentation.

Naturally, before wide-spread use, further animal studies and clinicaltrials will be conducted. The various elements of conducting a clinicaltrial, including patient treatment and monitoring, will be known tothose of skill in the art in light of the present disclosure. Thefollowing information is being presented as a general guideline for usein establishing such trials.

It is contemplated that patients chosen for combined studies would havefailed to respond to at least one course of conventional therapy and hadto have objectively measurable disease as determined by physicalexamination, laboratory techniques, or radiographic procedures. Wheremurine monoclonal antibody portions are employed in the immunotoxins orcoaguligands, the patients should have no history of allergy to mouseimmunoglobulin. Any chemotherapy should be stopped at least 2 weeksbefore entry into the study.

In regard to administration of the Tissue Factor constructs with eitherimmunotoxins or coaguligands, it is considered that certain advantageswill be found in the use of an indwelling central venous catheter with atriple lumen port. The therapeutic mixtures should be filtered, forexample, using a 0.22μ filter, and diluted appropriately, such as withsaline, to a final volume of 100 ml. Before use, the test sample shouldalso be filtered in a similar manner, and its concentration assessedbefore and after filtration by determining the A₂₈₀. The expectedrecovery should be within the range of 87 to 99%, and adjustments forprotein loss can then be accounted for.

These Tissue Factor and IT or coaguligand combinations may beadministered over a period of approximately 4-24 hours, with eachpatient receiving 2-4 infusions at 2-7 day intervals. Administration canalso bc performed by a steady rate of infusion over a 7 day period. Theinfusion given at any dose level should be dependent upon any toxicityobserved. Hence, if Grade II toxicity was reached after any singleinfusion, or at a particular period of time for a steady rate infusion,further doses should be withheld or the steady rate infusion stoppedunless toxicity improved. Increasing doses of Tissue Factor with eitherimmunotoxins or coaguligands should be administered to groups ofpatients until approximately 60% of patients showed unacceptable GradeIII or IV toxicity in any category. Doses that are 2/3 of this valuecould be defined as the safe dose.

Physical examination, tumor measurements, and laboratory tests should,of course, be performed before treatment and at intervals up to 1 monthlater. Laboratory tests should include complete blood counts, serumcreatinine, creatine kinase, electrolytes, urea, nitrogen, SGOT,bilirubin, albumin, and total serum protein. Serum samples taken up to60 days after treatment should be evaluated by radioimmunoassay for thepresence of the intact Tissue Factor, immunotoxin and/or coaguligand orcomponents thereof and antibodies against any portions thereof.Immunological analyses of sera, using any standard assay such as, forexample, an ELISA or RIA, will allow the pharmacokinetics and clearanceof the therapeutic agent to be evaluated.

To evaluate the anti-tumor responses, it is contemplated that thepatients should be examined at 48 hours to 1 week and again at 30 daysafter the last infusion. When palpable disease was present, twoperpendicular diameters of all masses should be measured daily duringtreatment, within 1 week after completion of therapy, and at 30 days. Tomeasure nonpalpable disease, serial CT scans could be performed at 1-cmintervals throughout the chest, abdomen, and pelvis at 48 hours to 1week and again at 30 days. Tissue samples should also be evaluatedhistologically, and/or by flow cytometry, using biopsies from thedisease sites or even blood or fluid samples if appropriate.

Clinical responses may be defined by acceptable measure. For example, acomplete response may be defined by the disappearance of all measurabletumor 1 month after treatment. Whereas a partial response may be definedby a 50% or greater reduction of the sum of the products ofperpendicular diameters of all evaluable tumor nodules 1 month aftertreatment, with no tumor sites showing enlargement. Similarly, a mixedresponse may be defined by a reduction of the product of perpendiculardiameters of all measurable lesions by 50% or greater 1 month aftertreatment, with progression in one or more sites.

F. Prolonged Half-Life TF

It is demonstrated herein that the anti-tumor activity of tTF isenhanced by conjugating tTF to carrier molecules, such asimmunoglobulins, that delay clearance of tTF from the body. For example,linking tTF to immunoglobulin enhances the anti-tumor activity byprolonging the in vivo half-life of tTF such that tTF persists forlonger and has more time to induce thrombotic events in tumor vessels.The prolongation in half-life either results from the increase in sizeof tTF above the threshold for glomerular filtration; or from activereadsorption of the conjugate within the kidney, a property of the Fcpiece of immunoglobulin (Spiegelberg and Weigle, 1965). It is alsopossible that the immunoglobulin component changes the conformation oftTF to render it more active or stable. Other carrier molecules besidesimmunoglobulin are contemplated to produce similar effects and are thusencompassed within the present invention.

F1. Modifications

Given that a first interpretation of the prolonged half-life observedupon the linkage of tTF to immunoglobulin is simply that the resultantincrease in size leads to prolonged plasma half-life, the inventorscontemplate that other modifications that increase the size of TFconstructs can be advantageously used in connection with the presentinvention, so long as the lengthening modification does notsubstantially restore membrane-binding functionality to the modified TFconstruct. Absent such a possibility, which can be readily tested,virtually any generally inert biologically acceptable molecule may beconjugated with a TF construct in order to prepare a modified TF withincreased in vivo half-life.

Modification may also be made to the structure of TF itself to render iteither more stable, or perhaps to reduce the rate of catabolism in thebody. One mechanism for such modifications is the use of d-amino acidsin place of l-amino acids in the TF molecule. Those of ordinary skill inthe art will understand that the introduction of such modificationsneeds to be followed by rigorous testing of the resultant molecule toensure that it still retains the desired biological properties. Furtherstabilizing modifications include the use of the addition of stabilizingmoieties to either the N-terminal or the C-terminal, or both, which isgenerally used to prolong the half-life of biological molecules. By wayof example only, one may wish to modify the termini of the TF constructsby acylation or amination. The variety of such modifications may also beemployed together, and portions of the TF molecule may also be replacedby peptidomimetic chemical structures that result in the maintenance ofbiological function and yet improve the stability of the molecule.

F2. Conjugates

i. Proteins

Techniques useful in connection with conjugation proteins of interest tocarrier proteins are widely used in the scientific community. It will begenerally understood that in the preparation of such TF conjugates foruse in the present invention, the protein chosen as a carrier moleculeshould have certain defined properties. For example, it must of coursebe biologically compatible and not result in any significant untowardeffects upon administration to a patient. Furthermore, it is generallyrequired that the carrier protein be relatively inert, andnon-immunogenic, both of which properties will result in the maintenanceof TF function and will allow the resultant construct to avoid excretionthrough the kidney. Exemplary proteins are albumins and globulins.

ii. Non Proteins

In common with the protein conjugates described above, the TF moleculesof the present invention may also be conjugated to non-protein elementsin order to improve their half-life in vivo. Again, the choice ofnon-protein molecules for use in such conjugates will be readilyapparent to those of ordinary skill in the art. For example, one may useany one or more of a variety of natural or synthetic polymers, includingpolysaccharides and PEG.

In the context of preparing conjugates, whether proteinaceous ornon-proteinaceous, one should take care that the introduced conjugatedoes not substantially reassociate the modified TF molecule with theplasma membrane such that it increases its coagulation ability andresults in a molecule that exerts harmful side effects followingadministration. As a general rule, it is believed that hydrophobicadditions or conjugates should largely be avoided in connection withthese embodiments.

iii. Immunoconjugates

Where antibodies are used to conjugate to the tTF compositions of thepresent invention, the choice of antibody will generally be dependent onthe intended use of the TF-antibody conjugate. For example, where the TFimmunoconjugates are contemplated for use in addition to the TFmolecules alone, the type of tumor should be considered, e.g., whetherit is preferable to target the tumor cells, or more preferably, thetumor vasculature or tumor stroma. Where the TF immunoconjugates arethemselves the primary therapeutic agents, the immunoconjugates will notin any sense be a "targeted immunoconjugate". In these aspects, theconjugation of the TF molecule to an antibody or portion thereof issimply performed in order to generate a construct that has improvedhalf-life and/or bioavailability in comparison to the original TFmolecule. In any event, certain advantages may be achieved through theapplication of particular types of antibodies. For example, while IgGbased antibodies may be expected to exhibit better binding capabilityand slower blood clearance than their Fab' counterparts, Fab'fragment-based compositions will generally exhibit better tissuepenetrating capability.

(a) Monoclonal Antibodies

Means for preparing and characterizing antibodies are well known in theart (See, e.g., Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory, 1988).

The methods for generating monoclonal antibodies (MAbs) generally beginalong the same lines as those for preparing polyclonal antibodies.Briefly, a polyclonal antibody is prepared by immunizing an animal withan immunogenic composition in accordance with the present invention,either with or without prior immunotolerizing, depending on the antigencomposition and protocol being employed (e.g., tolerizing to a normalcell population and then immunizing with a tumor cell population), andcollecting antisera from that immunized animal. A wide range of animalspecies can be used for the production of antisera. Typically the animalused for production of anti-antisera is a rabbit, a mouse, a rat, ahamster, a guinea pig or a goat. Because of the relatively large bloodvolume of rabbits, a rabbit is a preferred choice for production ofpolyclonal antibodies.

As is well known in the art, a given composition may vary in itsimmunogenicity. It is often necessary therefore to boost the host immunesystem, as may be achieved by coupling a peptide or polypeptideimmunogen to a carrier. Exemplary and preferred carriers are keyholelimpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albuminssuch as ovalbumin, mouse serum albumin or rabbit serum albumin can alsobe used as carriers. Means for conjugating a polypeptide to a carrierprotein are well known in the art and include glutaraldehyde,m-maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimyde andbis-biazotized benzidine.

As is also well known in the art, the immunogenicity of a particularimmunogen composition can be enhanced by the use of non-specificstimulators of the immune response, known as adjuvants. Exemplary andpreferred adjuvants include complete Freund's adjuvant (a non-specificstimulator of the immune response containing killed Mycobacteriumtuberculosis), incomplete Freund's adjuvants and aluminum hydroxideadjuvant.

The amount of immunogen composition used in the production of polyclonalantibodies varies upon the nature of the immunogen as well as the animalused for immunization. A variety of routes can be used to administer theimmunogen (subcutaneous, intramuscular, intradermal, intravenous andintraperitoneal). The production of polyclonal antibodies may bemonitored by sampling blood of the immunized animal at various pointsfollowing immunization. A second, booster injection, may also be given.The process of boosting and titering is repeated until a suitable titeris achieved. When a desired titer level is obtained, the immunizedanimal can be bled and the serum isolated and stored, and/or the animalcan be used to generate MAbs.

MAbs may be readily prepared through use of well-known techniques, suchas those exemplified in U.S. Pat. No. 4,196,265, incorporated herein byreference. Typically, this technique involves immunizing a suitableanimal with a selected immunogen composition, e.g., a purified orpartially purified tumor cell or vascular endothelial cell protein,polypeptide, peptide, or intact cell composition. The immunizingcomposition is administered in a manner effective to stimulate antibodyproducing cells. Rodents such as mice and rats are preferred animals,however, the use of rabbit, sheep frog cells is also possible. The useof rats may provide certain advantages (Goding, 1986, pp. 60-61), butmice are preferred, with the BALB/c mouse being most preferred as thisis most routinely used and generally gives a higher percentage of stablefusions.

Following immunization, somatic cells with the potential for producingantibodies, specifically B lymphocytes (B cells), are selected for usein the MAb generating protocol. These cells may be obtained frombiopsied spleens, tonsils or lymph nodes, or from a peripheral bloodsample. Spleen cells and peripheral blood cells are preferred, theformer because they are a rich source of antibody-producing cells thatare in the dividing plasmablast stage, and the latter because peripheralblood is easily accessible. Often, a panel of animals will have beenimmunized and the spleen of animal with the highest antibody titer willbe removed and the spleen lymphocytes obtained by homogenizing thespleen with a syringe. Typically, a spleen from an immunized mousecontains approximately 5×10⁷ to 2×10⁸ lymphocytes.

The antibody-producing B lymphocytes from the immunized animal are thenfused with cells of an immortal myeloma cell, generally one of the samespecies as the animal that was immunized. Myeloma cell lines suited foruse in hybridoma-producing fusion procedures preferably arenon-antibody-producing, have high fusion efficiency, and enzymedeficiencies that render then incapable of growing in certain selectivemedia which support the growth of only the desired fused cells(hybridomas).

Any one of a number of myeloma cells may be used, as are known to thoseof skill in the art (Goding, pp. 65-66, 1986;Campbell, pp. 75-83, 1984).For example, where the immunized animal is a mouse, one may useP3-X63/Ag8, X63-Ag8.653, NS1/1.Ag 4 1, Sp210-Ag14, FO, NSO/U, MPC-11,MPC11-X45-GTG 1.7 and S194/5XX0 Bul; for rats, one may use R210.RCY3,Y3-Ag 1.2.3, IR983F, 4B210 or one of the above listed mouse cell lines;and U-266, GM1500-GRG2, LICR-LON-HMy2 and UC729-6, are all useful inconnection with human cell fusions.

Methods for generating hybrids of antibody-producing spleen or lymphnode cells and myeloma cells usually comprise mixing somatic cells withmyeloma cells in a 4:1 proportion, though the proportion may vary fromabout 20:1 to about 1:1, respectively, in the presence of an agent oragents (chemical or electrical) that promote the fusion of cellmembranes. Fusion methods using Sendai virus have been described byKohler and Milstein (1975; 1976), and those using polyethylene glycol(PEG), such as 37% (v/v) PEG, by Gefter et al. (1977). The use ofelectrically induced fusion methods is also appropriate (Goding pp.71-74, 1986).

Fusion procedures usually produce viable hybrids at low frequencies,about 1×10⁻⁶ to 1×10⁻⁸. However, this does not pose a problem, as theviable, fused hybrids are differentiated from the parental, unfusedcells (particularly the unfused myeloma cells that would normallycontinue to divide indefinitely) by culturing in a selective medium. Theselective medium is generally one that contains an agent that blocks thede novo synthesis of nucleotides in the tissue culture media. Exemplaryand preferred agents are aminopterin, methotrexate, and azaserine.Aminopterin and methotrexate block de novo synthesis of both purines andpyrimidines, whereas azaserine blocks only purine synthesis. Whereaminopterin or methotrexate is used, the media is supplemented withhypoxanthine and thymidine as a source of nucleotides (HAT medium).Where azaserine is used, the media is supplemented with hypoxanthine.

The preferred selection medium is HAT. Only cells capable of operatingnucleotide salvage pathways are able to survive in HAT medium. Themyeloma cells are defective in key enzymes of the salvage pathway, e.g.,hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive.The B cells can operate this pathway, but they have a limited life spanin culture and generally die within about two weeks. Therefore, the onlycells that can survive in the selective media are those hybrids formedfrom myeloma and B cells.

This culturing provides a population of hybridomas from which specifichybridomas are selected. Typically, selection of hybridomas is performedby culturing the cells by single-clone dilution in microtiter plates,followed by testing the individual clonal supernatants (after about twoto three weeks) for the desired reactivity. The assay should besensitive, simple and rapid, such as radioimmunoassays, enzymeimmunoassays, cytotoxicity assays, plaque assays, dot immunobindingassays, and the like.

The selected hybridomas would then be serially diluted and cloned intoindividual antibody-producing cell lines, which clones can then bepropagated indefinitely to provide MAbs. The cell lines may be exploitedfor MAb production in two basic ways. A sample of the hybridoma can beinjected (often into the peritoneal cavity) into a histocompatibleanimal of the type that was used to provide the somatic and myelomacells for the original fusion. The injected animal develops tumorssecreting the specific monoclonal antibody produced by the fused cellhybrid. The body fluids of the animal, such as serum or ascites fluid,can then be tapped to provide MAbs in high concentration. The individualcell lines could also be cultured in vitro, where the MAbs are naturallysecreted into the culture medium from which they can be readily obtainedin high concentrations. MAbs produced by either means may be furtherpurified, if desired, using filtration, centrifugation and variouschromatographic methods such as HPLC or affinity chromatography.

The inventors also contemplate the use of a molecular cloning approachto generate monoclonals. For this, combinatorial immunoglobulin phagemidlibraries are prepared from RNA isolated from the spleen of theimmunized animal, and phagemids expressing appropriate antibodies areselected by panning using cells expressing the antigen and control cellse.g., normal-versus-tumor cells. The advantages of this approach overconventional hybridoma techniques are that approximately 10⁴ times asmany antibodies can be produced and screened in a single round, and thatnew specificities are generated by H and L chain combination whichfurther increases the chance of finding appropriate antibodies.

Where MAbs are employed in the present invention, they may be of human,murine, monkey, rat, hamster, chicken or even rabbit origin. Theinvention contemplates the use of human antibodies, "humanized" orchimeric antibodies from mouse, rat, or other species, bearing humanconstant and/or variable region domains, and other recombinantantibodies and fragments thereof. Of course, due to the ease ofpreparation and ready availability of reagents, murine monoclonalantibodies will typically be preferred.

(b) Functional Antibody Binding Regions Fab

Fab fragments can be obtained by proteolysis of the whole immunoglobulinby the non-specific thiol protease, papain. Papain must first beactivated by reducing the sulphydryl group in the active site withcysteine, 2-mercaptoethanol or dithiothreitol. Heavy metals in the stockenzyme should be removed by chelation with EDTA (2 mM) to ensure maximumenzyme activity. Enzyme and substrate are normally mixed together in theratio of 1:100 by weight. After incubation, the reaction can be stoppedby irreversible alkylation of the thiol group with iodoacetamide orsimply by dialysis. The completeness of the digestion should bemonitored by SDS-PAGE and the various fractions separated by proteinA-Sepharose or ion exchange chromatography.

F(ab')₂

The usual procedure for preparation of F(ab')₂ fragments from IgG ofrabbit and human origin is limited proteolysis by the enzyme pepsin(Protocol 7.3.2). The conditions, 100× antibody excess w/w in acetatebuffer at pH 4.5, 37° C., suggest that antibody is cleaved at theC-terminal side of the inter-heavy-chain disulfide bond. Rates ofdigestion of mouse IgG may vary with subclass and it may be difficult toobtain high yields of active F(ab')₂ fragments without some undigestedor completely degraded IgG. In particular, IgG_(2b) is highlysusceptible to complete degradation. The other subclasses requiredifferent incubation conditions to produce optimal results.

Digestion of rat IgG by pepsin requires conditions including dialysis in0.1 M acetate buffer, pH 4.5, and then incubation for four hours with 1%w/w pepsin; IgG₁ and IgG_(2a) digestion is improved if first dialyzedagainst 0.1 M formate buffer, pH 2.8, at 4° C., for 16 hours followed byacetate buffer. IgG_(2b) gives more consistent results with incubationin staphylococcal V8 protease (3% w/w) in 0.1 M sodium phosphate buffer,pH 7.8, for four hours at 37° C.

iv. Second Generation TF Immunoconjugates

The inventors contemplate that the Fc portion of the immunoglobulin inthe tTF-immunoglobulin construct employed in the advantageous studiesdisclosed herein may actually be the relevant portion of the antibodymolecule, resulting in increased in vivo half-life. It is reasonable topresume that the conjugation to the Fc region results in activereadsorption of a TF-Fc conjugate within the kidney, restoring theconjugate to the systemic circulation. As such, one may conjugate any ofthe coagulation-deficient TF constructs or variants of the invention toan Fc region in order to increase the in vivo half-life of the resultantconjugate.

Various methods are available for producing Fc regions in sufficientpurity to enable their conjugation to the TF constructs. By way ofexample only, the chemical cleavage of antibodies to provide the defineddomains or portions is well known and easily practiced, and recombinanttechnology can also be employed to prepare either substantial quantitiesof Fc regions or, indeed, to prepare the entire TF-Fc conjugatefollowing generation of a recombinant vector that expresses the desiredfusion protein.

Further manipulations of the general immunoglobulin structure may alsobe conducted with a view to providing second generation TF constructswith increased half-life. By way of example only, one may considerreplacing the C_(H) 3 domain of an IgG molecule with a truncated TissueFactor or variant thereof. In general, the most effective mechanism forproducing such a hybrid molecule will be to use molecular cloningtechniques and recombinant expression. All such techniques are generallyknown to those of ordinary skill in the art, and are further describedin detail herein.

F3. Linkage Means

The compositions above may be linked to the Tissue Factor compositionsin any operative manner that allows each region to perform its intendedfunction without significant impairment of the Tissue Factor functions.Thus, the linking components will be capable prolonging the half life ofthe construct, and the Tissue Factor is capable of promoting bloodcoagulation or clotting.

i. Biochemical Cross-Linkers

The joining of any of the above components, to a Tissue Factorcomposition will generally employ the same technology as developed forthe preparation of immunotoxins. It can be considered as a generalguideline that any biochemical cross-linker that is appropriate for usein an immunotoxin will also be of use in the present context, andadditional linkers may also be considered.

Cross-linking reagents are used to form molecular bridges that tietogether functional groups of two different molecules, e.g., astabilizing and coagulating agent. To link two different proteins in astep-wise manner, hetero-bifunctional cross-linkers can be used thateliminate unwanted homopolymer formation.

                                      TABLE IV                                    __________________________________________________________________________    HETERO-BIFUNCTIONAL CROSS-LINKERS                                                                             Spacer Arm                                                                    Length\after cross-                 linker  Reactive Toward                                                                       Advantages and Applications                                                                   linking                                       __________________________________________________________________________    SMPT    Primary amines                                                                        . Greater stability                                                                           11.2 A                                                Sulfhydryls                                                           SPDP    Primary amines                                                                        . Thiolation     6.8 A                                                Sulfhydryls                                                                           . Cleavable cross-linking                                     LC-SPDP Primary amines                                                                        . Extended spacer arm                                                                         15.6 A                                                Sulfhydryls                                                           Sulfo-LC-SPDP                                                                         Primary amines                                                                        . Extended spacer arm                                                                         15.6 A                                                Sulfydryls                                                                            . Water-soluble                                               SMCC    Primary amines                                                                        . Stable maleimide reactive group                                                             11.6 A                                                Sulfhydryls                                                                           . Enzyme-antibody conjugation                                                 . Hapten-carrier protein conjugation                          Sulfo-SMCC                                                                            Primary amines                                                                        . Stable maleimide reactive group                                                             11.6 A                                                Sulfhydryls                                                                           . Water-soluble                                                               . Enzyme-antibody conjugation                                 MBS     Primary amines                                                                        . Enzyme-antibody conjugation                                                                  9.9 A                                                Sulfhydryls                                                                           . Hapten-carrier protein conjugation                          Sulfo-MBS                                                                             Primary amines                                                                        . Water-soluble  9.9 A                                                Sulfhydryls                                                           SIAB    Primary amines                                                                        . Enzyme-antibody conjugation                                                                 10.6 A                                                Sulfhydryls                                                           Sulfo-SIAB                                                                            Primary amines                                                                        . Water-soluble 10.6 A                                                Sulfhydryls                                                           SMPB    Primary amines                                                                        . Extended spacer arm                                                                         14.5 A                                                Sulfhydryls                                                                           . Enzyme-antibody conjugation                                 Sulfo-SMPB                                                                            Primary amines                                                                        . Extended spacer arm                                                                         14.5 A                                                Sulfhydryls                                                                           . Water-soluble                                               EDC/Sulfo-NHS                                                                         Primary amines                                                                        . Hapten-Carrier conjugation                                                                  0                                                     Carboxyl groups                                                       ABH     Carbohydrates                                                                         . Reacts with sugar groups                                                                    11.9 A                                                Nonselective                                                          __________________________________________________________________________

An exemplary hetero-bifunctional cross-linker contains two reactivegroups: one reacting with primary amine group (e.g., N-hydroxysuccinimide) and the other reacting with a thiol group (e.g., pyridyldisulfide, maleimides, halogens, etc.). Through the primary aminereactive group, the cross-linker may react with the lysine residue(s) ofone protein (e.g., the selected antibody or fragment) and through thethiol reactive group, the cross-linker, already tied up to the firstprotein, reacts with the cysteine residue (free sulfhydryl group) of theother protein (e.g., the coagulant).

It can therefore be seen that the preferred Tissue Factor compositionwill generally have, or be derivatized to have, a functional groupavailable for cross-linking purposes. This requirement is not consideredto be limiting in that a wide variety of groups can be used in thismanner. For example, primary or secondary amine groups, hydrazide orhydrazine groups, carboxyl alcohol, phosphate, or alkylating groups maybe used for binding or cross-linking. For a general overview of linkingtechnology, one may wish to refer to Ghose and Blair (1987).

The spacer arm between the two reactive groups of a cross-linkers mayhave various length and chemical compositions. A longer spacer armallows a better flexibility of the conjugate components while someparticular components in the bridge (e.g., benzene group) may lend extrastability to the reactive group or an increased resistance of thechemical link to the action of various aspects (e.g., disulfide bondresistant to reducing agents). The use of peptide spacers, such asL-Leu-L-Ala-L-Leu-L-Ala, is also contemplated.

It is preferred that a cross-linker having reasonable stability in bloodwill be employed. Numerous types of disulfide-bond containing linkersare known that can be successfully employed to conjugate targeting andcoagulating agents. Linkers that contain a disulfide bond that issterically hindered may prove to give greater stability in vivo,preventing release of the Tissue Factor prior to binding at the site ofaction. These linkers are thus one preferred group of linking agents.

One of the most preferred cross-linking reagents for use in immunotoxinsis SMPT, which is a bifunctional cross-linker containing a disulfidebond that is "sterically hindered" by an adjacent benzene ring andmethyl groups. It is believed that steric hindrance of the disulfidebond serves a function of protecting the bond from attack by thiolateanions such as glutathione which can be present in tissues and blood,and thereby help in preventing decoupling of the conjugate prior to thedelivery of the attached agent to the tumor site. It is contemplatedthat the SMPT agent may also be used in connection with the bispecificcoagulating ligands of this invention.

The SMPT cross-linking reagent, as with many other known cross-linkingreagents, lends the ability to cross-link functional groups such as theSH of cysteine or primary amines (e.g., the epsilon amino group oflysine). Another possible type of cross-linker includes thehetero-bifunctional photoreactive phenylazides containing a cleavabledisulfide bond such as sulfosuccinimidyl-2-(p-azido salicylamido)ethyl-1,3'-dithiopropionate. The N-hydroxy-succinimidyl group reactswith primary amino groups and the phenylazide (upon photolysis) reactsnon-selectively with any amino acid residue.

In addition to hindered cross-linkers, non-hindered linkers can also beemployed in accordance herewith. Other useful cross-linkers, notconsidered to contain or generate a protected disulfide, include SATA,SPDP and 2-iminothiolane (Wawrzynczak and Thorpe, 1987). The use of suchcross-linkers is well understood in the art.

Once conjugated, the tTF will generally be purified to separate theconjugate from unconjugated targeting agents or coagulants and fromother contaminants. A large a number of purification techniques areavailable for use in providing conjugates of a sufficient degree ofpurity to render them clinically useful. Purification methods based uponsize separation, such as gel filtration, gel permeation or highperformance liquid chromatography, will generally be of most use. Otherchromatographic techniques, such as Blue-Sepharose separation, may alsobe used.

ii. Recombinant Fusion Proteins

The tTF compositions of the invention may also be fusion proteinsprepared by molecular biological techniques. The use of recombinant DNAtechniques to achieve such ends is now standard practice to those ofskill in the art. These methods include, for example, in vitrorecombinant DNA techniques, synthetic techniques and in vivorecombination/genetic recombination. DNA and RNA synthesis may,additionally, be performed using an automated synthesizers (see, forexample, the techniques described in Sambrook et al., 1989;and Ausubelet al., 1989).

The preparation of such a fusion protein generally entails thepreparation of a first and second DNA coding region and the functionalligation or joining of said regions, in frame, to prepare a singlecoding region that encodes the desired fusion protein. In the presentcontext, the tTF or TF mutant DNA sequence will generally be joined inframe with a DNA sequence encoding an inert protein carrier,immunoglobulin, Fc region, or such like. It is not generally believed tobe particularly relevant whether the TF portion of the fusion protein orthe inert portion is prepared as the N-terminal region or as theC-terminal region. In connection with the second generation TFimmunoglobulin molecules, the TF coding sequences may further beinserted within the immunoglobulin coding regions, such that the TFsequences functionally interrupt the immunoglobulin sequences and theencoded protein may be considered a "tribrid".

Once the coding region desired has been produced, an expression vectoris created. Expression vectors contain one or more promoters upstream ofthe inserted DNA regions that act to promote transcription of the DNAand to thus promote expression of the encoded recombinant protein. Thisis the meaning of "recombinant expression" and has been discussedelsewhere in the specification.

F4. Assays

As with other aspects of the present invention, once a candidate TFconstruct has been generated with the intention of providing a constructwith increased in vivo half-life, the construct should generally betested to ensure that the desired properties have been imparted to theresultant compound. The various assays for use in determining suchchanges in function are routine and easily practiced by those ofordinary skill in the art.

In TF conjugates designed simply in order to increase their size,confirmation of increased size is completely routine. For example, onewill simply separate the candidate composition using any methodologythat is designed to separate biological components on the basis of sizeand one will analyze the separated products in order to determine that aTF construct of increased size has been generated. By way of exampleonly, one may mention separation gels and separation columns, such asgel filtration columns. The use of gel filtration columns in theseparation of mixtures of conjugated and non-conjugated components mayalso be useful in other aspects of the present invention, such as in thegeneration of relatively high levels of conjugates, immunotoxins orcoaguligands.

As the objective of the present class of conjugates is to provide acoagulation-deficient TF molecule having an increased in vivo half-life,the candidate TF modified variants or conjugates should generally betested in order to confirm that this property is present. Again, suchassays are routine in the art. A first simple assay would be todetermine the half-life of the candidate modified or conjugated TF in anin vitro assay. Such assays generally comprise mixing the candidatemolecule in sera and determining whether or not the molecule persists ina relatively intact form for a longer period of time, as compared to theinitial sample of coagulation-deficient Tissue Factor. One would againsample aliquots from the admixture and determine their size, andpreferably, their biological function.

In vivo assays of biological half-life or "clearance" can also be easilyconducted. In these systems, it is generally preferred to label the testcandidate TF constructs with a detectable marker and to follow thepresence of the marker after administration to the animal, preferablyvia the route intended in the ultimate therapeutic treatment strategy.As part of this process, one would take samples of body fluids,particularly serum and/or urine samples, and one would analyze thesamples for the presence of the marker associated with the TF construct,which will indicate the longevity of the construct in the naturalenvironment in the body.

Any one or more or a combination of the TF molecules with increasedhalf-life may thus be used in conjunction with the therapeutic methodsdisclosed herein. The doses proposed for administration will generallybe between about not 0.2 mg and about 200 mg per patient, as with theoriginal TF constructs described above. However, in that these TFmolecules have been modified, it is possible that the effective dosesmay be even lower, such as on the order of about not 0.1 mg. It is morelikely that the therapeutic treatment regimens will be altered whenusing the increased half-life TFs in the number of times that thepharmaceuticals are administered, rather than in alteration of the givendoses. For example, where an original TF construct is proposed for useon days 1, 3 and 7 within the treatment period, the counterpart improvedTF with longer half-life may rather be administered only on day 1 andday 7. In any event, all such optimizations in terms of doses and timesfor administration will be easily determined by those of ordinary skillin the art.

G. TF and Factor VIIa Combinations

The inventors have further demonstrated that coagulation-inducingactivity of tTF bound to A20 cells was markedly enhanced in the presenceof Factor VIIa. In common with earlier studies, these in vitro resultsalso translated to the in vivo environment. Studies are presented hereinto demonstrate that the anti-tumor activity of variouscoagulation-deficient TF constructs is enhanced upon co-administrationwith Factor VIIa. Even using an experimental animal model of the HT29tumor, which is notoriously difficult to coagulate, theco-administration of coagulation-deficient TF constructs and exogenousFactor VIIa resulted in considerable necrosis of the tumor tissue.

This data can be explained as tTF binds Factor VII but does notefficiently mediate its activation to Factor VIIa by Xa and adjacentFactor VIIa molecules. Providing a source of preformed (exogenous)Factor VIIa overcomes this block, enabling more efficient coagulation.The success of the combined coagulation-deficient TF and Factor VIIatreatment is generally based upon the surprising localization of the TFconstruct within the vasculature of the tumor. Absent such surprisinglocalization and specific functional effects, the co-administration ofFactor VIIa would not be meaningful in the context of tumor treatment,and may even be harmful as it may promote unwanted thrombosis in varioushealthy tissues. The combined use of tTF and Factor VIIa in anon-targeted manner has previously been proposed in connection with thetreatment of hemophiliacs and patients with other bleeding disorders, inwhich there is a fundamental impairment of the coagulation cascade. Inthe present invention, the coagulation cascade is generally fullyoperative, and the therapeutic intervention concentrates this activitywithin a defined region of the body.

It is therefore a further object of the present invention to increasethe anti-tumor effects of any one of the TF constructs of the inventionby combining the use of TF with the additional administration of FactorVIIa. As tTF binds to tumor vascular endothelium, it is possible toinject tTF into tumor-bearing animals, wait a period of time for excesstTF to be cleared, and then inject Factor VIIa to magnify the thromboticaction of the tTF within tumor vessels. In this manner, the tTF or othercoagulation-deficient TF construct can be seen to form a reservoirwithin the tumor, allowing the subsequent administration of Factor VIIato increase and perpetuate the anti-tumor effect.

A further observation of the present invention is that the thromboticactivity of the Factor VII activation mutants of tTF (G164A) and tTF(W158R) was largely restored by Factor VIIa. These mutations lie withina region of tTF that is important for the conversion of Factor VII toFactor VIIa. As with tTF itself, the studies herein show that addingpreformed Factor VIIa overcomes this block in coagulation complexformation. The present invention exploits these and the aforementionedobservations with a view to providing in vivo therapy of cancer.

Indeed, the studies presented herein confirm that the co-administrationof a Factor VII activation mutant variant of TF with preformed FactorVIIa results in considerable necrotic damage to the tumors, even insmall tumor models which are not the most amenable to treatment with thepresent invention. This aspect of the invention is particularlysurprising as it was not previously believed that such mutants wouldhave any therapeutic utility in any embodiments other than, perhaps, inthe competitive inhibition of TF as may be used to inhibit or reducecoagulation. Apart from such hypotheses, the generation of such mutantshas been motivated by scientific interest and they could perhaps be usedas controls in certain in vitro studies. Only the studies of the presentinventors render such mutants clinically useful, either in the contextof targeted delivery (WO 96/01653), or in the even more surprisingcombined uses of the present invention.

In particular embodiments, this application of the present inventiontherefore first involves injecting tTF (G164A), tTF (W158R) or anequivalent thereof into tumor bearing animals. The tTF mutant is thenallowed to localize to tumor vessels and the residue is cleared. This isthen followed by the injection of Factor VIIa, which allows thelocalized tTF mutants to express thrombotic activity. This strategyoffers the advantage that it is very safe. The tTF mutants arepractically non-toxic, as is Factor VIIa itself. Thus, administering thetTF mutant followed by Factor VIIa will be harmless to the host, yetefficiently induce thrombosis of tumor vessels.

G1. Factor VIIa

Factor VII can be prepared as described by Fair (1983), and as shown inU.S. Pat. Nos. 5,374,617, 5,504,064 and 5,504,067, each of which isincorporated herein by reference. The coding portion of the human FactorVII cDNA sequence was reported by Hagen et al., (1986). The amino acidsequence from 1 to 60 corresponds to the pre-pro/leader sequence that isremoved by the cell prior to secretion. The mature Factor VIIpolypeptide chain consists of amino acids 61 to 466. Factor VII isconverted to its active form, Factor VIIa, by cleavage of a singlepeptide bond between arginine-212 and isoleucine-213.

Factor VII can be converted in vitro to Factor VIIa by incubation of thepurified protein with Factor Xa immobilized on Affi-Gel™ 15 beads(Bio-Rad). Conversion can be monitored by SDS-polyacrylamide gelelectrophoresis of reduced samples. Free Factor Xa in the Factor VIIapreparation can be detected with the chromogenic substratemethoxycarbonyl-D-cyclohexylglycyl-glycyl-arginine-p-nitroanilideacetate (Spectrozyme™ Factor Xa, American Diagnostica, Greenwich, Conn.)at 0.2 mM final concentration in the presence of 50 mM EDTA.

Recombinant Factor VIIa can also be purchased from Novo Biolabs(Danbury, Conn.).

G2. Treatment

The use of Factor VIIa in connection with the present invention is notconfined to its ability to significantly improve the utility of theFactor VII activation mutants disclosed herein. It is equallycontemplated that Factor VIIa will be used in conjunction with thecoagulation-deficient Tissue Factor molecules of equivalent activity tothe truncated tTF first employed. In such treatment embodiments, thedose of the TF construct will generally be between about not 0.2 mg andabout 200 mg per patient. The appropriate doses of Factor VIIa can bestbe determined in light of this information.

For example, it may be desired to create a 1:1 ratio of the TF constructand Factor VIIa in a precomplex and to administer the precomplexedcomposition to the animal. Should this be desired, one would generallyadmix an amount of TF and an amount of Factor VIIa sufficient to allowthe formation of an equimolar complex. To achieve this, it may bepreferable to use a 2-3 molar excess of Factor VIIa in order to ensurethat each of the TF molecules are adequately complexed. One would thensimply separate the uncomplexed TF and Factor VIIa from the complexedmixture using any suitable technique, such as gel filtration. Afterformation of the TF:VIIa complex, one may simply administer the complexto a patient in need of treatment in a dose of between about not 0.2 mgand about 200 mg per patient.

As stated above, it may generally be preferred to administer thecoagulation-deficient TF construct to a patient in advance, allowing theTF sufficient time to localize specifically within the tumor. Followingsuch preadministration, one would then design an appropriate dose ofFactor VIIa sufficient to coordinate and complex with the TF localizedwithin the tumor vasculature. Again, one may design the dose of FactorVIIa in order to allow a 1:1 molar ratio of TF and Factor VIIa to formin the tumor environment. Given the differences in molecular weight ofthese two molecules, it will be seen that it would be advisable to addapproximately twice the amount in milligrams of Factor VIIa incomparison to the milligrams of TF.

However, the foregoing analysis is merely exemplary, and any doses ofFactor VIIa that generally result in an improvement in coagulation wouldevidently be of clinical significance. In this regard, it is notablethat the studies presented herein in fact use a 16:1 excess of TF incomparison to Factor VIIa, which is generally about a 32-fold molarexcess of the TF construct. Nevertheless, impressive coagulation andnecrosis was specifically observed in the tumor. Therefore, it will beevident that the effective doses of Factor VIIa are quite broad. By wayof example only, one may consider administering to a patient a dose ofFactor VIIa between about 0.01 mg and about 500 mg per patient.

Each of the foregoing analyses may be equally applied to the use ofTissue Factor constructs that have been mutated to impair their abilityto activate Factor VII. Given that the foregoing calculations are basedupon a ratio of binding, it is not believed to be necessary to useparticularly increased levels of Factor VIIa in combination with theactivation mutants described. However, given that the administration ofFactor VlIa is not believed to be particularly harmful in itself, thepotential for using increased doses of Factor VIIa is certainly evident.

Although the detailed guidance provided above is believed to besufficient to enable one of ordinary skill in the art how to practicethese aspects of the invention, one may also refer to other quantitativeanalyses to assist in the optimization of the TF and Factor VIIa dosesfor administration. By way of example only, one may refer to U.S. Pat.Nos. 5,374,617; 5,504,064;and 5,504,067, which describe a range oftherapeutically active doses and plasma levels of Factor VIIa.

Morrissey and Comp have reported that, in the context of bleedingdisorders, the coagulation-deficient Tissue Factor may be administeredin a dosage effective to produce in the plasma an effective level ofbetween 100 ng/ml and 50 μg/ml, or a preferred level of between 1 μg/mland 10 μg/ml or 60 to 600 μg/kg body weight, when administeredsystemically; or an effective level of between 10 μg/ml and 50 μg/ml, ora preferred level of between 10 μg/ml and 50 μg/ml, when administeredtopically (U.S. Pat. No. 5,504,064).

The Factor VIIa is administered in a dosage effective to produce in theplasma an effective level of between 20 ng/ml and 10 μg/ml, (1.2 to 600μg/kg), or a preferred level of between 40 ng/ml and 700 μg/ml (2.4 to240 μg/kg), or a level of between 1 μg Factor VIIa/ml and 10 μg FactorVIIa/ml when administered topically.

In general, one would administer coagulation-deficient Tissue Factor andFactor VII activator to produce levels of up to 10 μgcoagulation-deficient Tissue Factor/ml plasma and between 40 ng and 700μg Factor VIIa/ml plasma. While these studies were performed in thecontext of bleeding disorders, they have also relevance in the contextof the present invention, in that levels must be effective butappropriately monitored to avoid systemic toxicity due to elevatedlevels of coagulation-deficient Tissue Factor and activated Factor VIIa.Therefore, the Factor VII activator is administered in a dosageeffective to produce in the plasma an effective level of Factor VIIa, asdefined above.

G3. Factor VII Activators

As described in U.S. Pat. No. 5,504,064, incorporated herein byreference, activators of endogenous Factor VII may also be administeredin place of Factor VIIa itself. As described in the foregoing patent,Factor VIIa can also be formed in vivo, shortly before, at the time of,or preferably slightly after the administration of thecoagulation-deficient Tissue Factors. In such embodiments, endogenousFactor VII is converted into Factor VIIa by infusion of an activator ofFactor VIIa, such as Factor Xa (FXa) in combination with phospholipid(PCPS).

Activators of Factor VII in vivo include Factor Xa/PCPS, FactorIXa/PCPS, thrombin, Factor XIIa, and the Factor VII activator from thevenom of Oxyuranus scutellatus in combination with PCPS. These have beenshown to activate Factor VII to Factor VIIa in vitro. Activation ofFactor VII to Factor VIIa for Xa/PCPS in vivo has also been measureddirectly. In general, the Factor VII activator is administered in adosage between 1 and 10 μg/ml of carrier (U.S. Pat. No. 5,504,064).

The phospholipid can be provided in a number of forms such asphosphatidyl choline/phosphatidyl serine vesicles (PCPS). The PCPSvesicle preparations and the method of administration of Xa/PCPS isdescribed in Giles et al., (1988), the teachings of which arespecifically incorporated herein. Other phospholipid preparations can besubstituted for PCPS, so long as they accelerate the activation ofFactor VII by Factor Xa. Effectiveness, and therefore determination ofoptimal composition and dose, can be monitored as described below.

A highly effective dose of Xa/PCPS, which elevates Factor VIIa levels invivo in the chimpanzee, has been reported to be 26 pmoles FXa+40 pmolesPCPS per kg body weight. That dose yielded an eighteen fold increase inendogenous levels of Factor VIIa (to 146 ng/ml). A marginally detectableeffect was observed using a smaller dose in dogs, where the infusion of12 pmoles Factor Xa+19 pmoles PCPS per kg body weight yielded a threefold increase in endogenous Factor VIIa levels. Accordingly, doses ofFactor Xa that are at least 12 pmoles Factor Xa per kg body weight, andpreferably 26 pmoles Factor Xa per kg body weight, should be useful.Doses of PCPS that are at least 19 pmoles PCPS per kg body weight, andpreferably 40 pmoles PCPS per kg body weight, are similarly useful (U.S.Pat. No. 5,504,064).

The effectiveness of any infusible Factor VII activator can bemonitored, following intravenous administration, by drawing citratedblood samples at varying times (at 2, 5, 10, 20, 30, 60, 90 and 120min.) following a bolus infusion of the activator, and preparingplatelet-poor plasma from the blood samples. The amount of endogenousFactor VIIa can then be measured in the citrated plasma samples byperforming a coagulation-deficient Tissue Factor-based Factor VIIaclotting assay. Desired levels of endogenous Factor VIIa would be thesame as the target levels of plasma Factor VIIa indicated forco-infusion of purified Factor VII and coagulation-deficient TissueFactor. Therefore, other activators of Factor VII could be tested invivo for generation of Factor VIIa, without undue experimentation, andthe dose adjusted to generate the desirable levels of Factor VIIa, usingthe coagulation-deficient Tissue Factor-based Factor VIIa assay ofplasma samples. The proper dose of the Factor VII activator (yieldingthe desired level of endogenous Factor VIIa) can then be used incombination with the recommended amounts of coagulation-deficient TissueFactor.

Doses can be timed to provide prolong elevation in Factor VIIa levels.Preferably doses would be administered until the desired anti-tumoreffect is achieved, and then repeated as needed to control bleeding. Thehalf-life of Factor VIIa in vivo has been reported to be approximatelytwo hours, although this could vary with different therapeuticmodalities and individual patients. Therefore, the half-life of FactorVIIa in the plasma in a given treatment modality should be determinedwith the coagulation-deficient Tissue Factor-based clotting assay.

H. Examples

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

EXAMPLE I Synthesis of Truncated Tissue Factor

tTF is herein designated as the extracellular domain of the matureTissue Factor protein (amino acid 1-219 of the mature protein; SEQ IDNO:1). SEQ ID NO:1 is encoded by, e.g., SEQ ID NO:10.

A. H₆ [tTF]

H₆ Ala Met Ala[tTF]. The tTF complimentary DNA (cDNA) was prepared asfollows: RNA from J-82 cells (human bladder carcinoma) was used for thecloning of tTF. Total RNA was isolated using the GlassMax™ RNAmicroisolation reagent (Gibco BRL). The RNA was reverse transcribed tocDNA using the GeneAmp RNA PCR kit (Perkin Elmer). tTF cDNA wasamplified using the same kit with the following two primers:

    ______________________________________                                        5' primer: 5' GTC ATG CCA TGG CCT CAG GCA CTA CAA                             (SEQ ID NO: 15)                                                               3' primer: 5' TGA CAA GCT TAT TCT CTG AAT TCC CCT TTC T                       (SEQ ID NO: 16)                                                               ______________________________________                                    

The underlined sequences codes for the N-terminus of tTF. The rest ofthe sequence in the 5' primer is the restriction site for NcoI allowingthe cloning of tTF into the expression vector. The sequence in the 3'primer is the HindIII site for cloning tTF into the expression vector.PCR amplification was performed as suggested by the manufacturer.Briefly, 75 μM dNTP;0.6 μM primer, 1.5 mM MgCl₂ were used and 30 cyclesof 30" at 95° C., 30" at 55° C. and 30" at 72° C. were performed.

The tTF was expressed as a fusion protein in a non-native state in E.coli inclusion bodies using the expression vector H₆ pQE-60 (Qiagen).The E. coli expression vector H₆ pQE-60 was used for expressing tTF (Leeet al., 1994). The PCR amplified tTF cDNA was inserted between the NcoIand HindIII site. H₆ pQE-60 has a built-in (His)₆ encoding sequence suchthat the expressed protein has the sequence of (His)₆ at the N terminus,which can be purified on a Ni-NTA column. In addition, the fusionprotein has a thrombin cleavage site and residues 1-219 of TF.

To purify tTF, tTF containing H6 pQE-60 DNA was transformed to E. coliTG-1 cells. The cells were grown to OD₆₀₀ =0.5 and IPTG was added to 30μM to induce the tTF production. The cells were harvested after shakingfor 18 h at 30° C. The cell pellet was denatured in 6 M Gu-HCl and thelysate was loaded onto a Ni-NTA column (Qiagen). The bound tTF waswashed with 6 M urea and tTF was refolded with a gradient of 6 M-1 Murea at room temperature for 16 h. The column was washed with washbuffer (0.05 Na H₂ PO₄, 0.3 M NaCl, 10% glycerol) and tTF was elutedwith 0.2 M Imidozole in wash buffer. The eluted tTF was concentrated andloaded onto a G-75 column. tTF monomers were collected.

B. tTF

Gly[tTF]. The GlytTF complimentary DNA (cDNA) was prepared the same wayas described in the previous section except the 5' primer was replacedby the following primer in the PCR.

    5' primer: 5' GTC ATG CCA TGG CCC TGG TGC CTC GTG CTT CTG GCA CTA CAA ATA CT(SEQ ID NO:17)

The underlined sequence codes for the N-terminus of tTF. The remainingsequence encodes a restriction site for NcoI and a cleavage site forthrombin.

The H₆ pQE60 expression vector and the procedure for proteinpurification is identical to that described above except that the finalprotein product was treated with thrombin to remove the H₆ peptide. Thiswas done by adding 1 part of thrombin (Sigma) to 500 parts of tTF (w/w),and the cleavage was carried out at room temperature for 18 h. Thrombinwas removed from tTF by passage of the mixture through a BenzamidineSepharose 6B thrombin affinity column (Pharmacia). The resultant tTF,designated tTF₂₁₉, consisted of residues 1-219 of TF plus an additionalglycine at the N-terminus. It migrated as a single band of molecularweight 26 kDa when analyzed by SDS-PAGE, and the N-terminal sequence wasconfirmed by Edman degradation. It has the sequence of SEQ ID NO: 1.

C. Cysteine-Modified tTFs

(His)₆ -N'-cys'tTF₂₁₉ -tTF, hereafter abbreviated to H₆ -N'-cys-tTF₂₁₉,was prepared by mutating tTF₂₁₉ by PCR with a 5' primer encoding a Cysin front of the N'-terminus of mature tTF. H₆ -tTF₂₁₉ -cys-C' wasprepared likewise using a 3' primer encoding a Cys after amino acid 219of tTF. Expression and purification were as for tTF₂₁₉ except thatEllman's reagent (5'5'-dithio-bis-2-nitrobenzoic acid) was applied afterrefolding to convert the N'- or C'-terminal Cys into a stable activateddisulfide group. The products have the sequences shown in SEQ ID NO:2and SEQ ID NO:3. Thrombin cleavage removed the (His)₆ tag and convertedthe proteins into N'-cys-tTF₂₁₉ and tTF₂₁₉ -cys-C' having the sequencesshown in SEQ ID NO:4 and SEQ ID NO:5. The products were >95% pure asjudged by SDS-polyacrylamide gel electrophoresis.

H₆ -tTF₂₂₀ -cys-C' and H₆ -tTF₂₂₁ -cys-C' were prepared by mutatingtTF₂₁₉ by PCR with 3' primers encoding Ile-Cys and Ile-Phe-Cys afteramino acid 219 of tTF. Expression, refolding and purification were asfor H₆ -tTF₂₁₉ -cys-C'. The proteins have the sequences shown in SEQ IDNO:6 and SEQ ID NO:7.

EXAMPLE II Synthesis of Dimeric Tissue Factor

The inventors' reasoned that Tissue Factor dimers may be more potentthan monomers at initiating coagulation. It is possible that nativeTissue Factor on the surface of J82 bladder carcinoma cells may exist asa dimer (Fair et al., 1987). The binding of one Factor VII or FactorVIIa molecule to one Tissue Factor molecule may also facilitate thebinding of another Factor VII or Factor VIIa to another Tissue Factor(Fair et al., 1987;Bach et al., 1986). Furthermore, Tissue Factor showsstructural homology to members of the cytokine receptor family(Edgington et al., 1991) some of which dimerize to form active receptors(Davies and Wlodawer, 1995). The inventors therefore synthesized TFdimers, as follows. While the synthesis of dimers hereinbelow isdescribed in terms of chemical conjugation, recombinant and other meansfor producing the dimers of the present invention are also contemplatedby the inventors.

A. [tTF] Linker [tTF]

The Gly [tTF] Linker [tTF] with the structure Gly[tTF] (Gly)₄ Ser (Gly)₄Ser (Gly)₄ Ser [tTF] was made. Two pieces of DNA were PCR amplifiedseparately and were ligated and inserted into the vector as follows:

PCR 1: Preparation of tTF and the 5' half of the linker DNA. The primersequences in the PCR are as follows:

    __________________________________________________________________________    5' primer: 5' GTC ATG CCA TGG CCC TGG TGC CTC GTG GTT CTT GCG GCA             CTA CAA ATA CT (SEQ ID NO: 18)                                                3' primer: 5' CGC GGA TCC ACC GCC ACC AGA TCC ACC GCC TCC TTC TCT             GAA TTC CCC TTT CT (SEQ ID NO: 19)                                            __________________________________________________________________________

Gly[tTF] DNA was used as the DNA template. Further PCR conditions wereas described in the tTF section.

PCR 2: Preparation of the 3' half of the linker DNA and tTF DNA. Theprimer sequences in the PCR were as follows:

    __________________________________________________________________________    5' primer: 5' CGC GGA TCC GGC GGT GGA GGC TCT TCA GGC ACT ACA AAT             ACT GT (SEQ ID NO: 20)                                                        3' primer: 5' TGA CAA GCT TAT TCT CTG AAT TCC CCT TTC T                       (SEQ ID NO: 21)                                                               __________________________________________________________________________

tTF DNA was used as the template in the PCR. The product from PCR 1 wasdigested with NcoI and BamH. The product from PCR 2 was digested withHindIII and BamH1. The digested PCR1 and PCR2 DNA were ligated with NcoIand HindIII-digested H₆ pQE 60 DNA.

For the vector constructs and protein purification, the procedures werethe same as described in the Gly [tTF] section.

B. Cys [tTF] Linker [tTF]

The Cys [tTF] Linker [tTF] with the structure Ser Gly Cys [tTF 2-219](Gly)₄ Ser (Gly)₄ Ser(Gly)₄ Ser [tTF] was also constructed. DNA was madeby PCR using the following primers were used:

    __________________________________________________________________________    5' primer: 5' GTC ATG CCA TGG CCC TGG TGC CTC GTG GTT CTT GCG GCA             CTA CAA ATA CT (SEQ ID NO: 22)                                                3' primer: 5' TGA CAA GCT TAT TCT CTG AAT TCC CCT TTC T                       (SEQ ID NO: 23)                                                               __________________________________________________________________________

[tTF] linker [tTF] DNA was used as the template. The remaining PCRconditions were the same as described in the tTF section. The vectorconstructs and protein purification were all as described in thepurification of H₆ C[tTF].

C. [tTF] Linker [tTF]cys

The [tTF] Linker [tTF]cys dimer with the protein structure [tTF] (Gly)₄Ser (Gly)₄ Ser (Gly)₄ Ser [tTF] Cys was also made. The DNA was made byPCR using the following primers:

    __________________________________________________________________________    5' primer: 5' GTC ATG CCA TGG CCC TGG TGC CTC GTG GTT GCA CTA CAA             ATA CT (SEQ ID NO: 24)                                                        3' primer: 5' TGA CAA GCT TAG CAT TCT CTG AAT TCC CCT TTC T (SEQ ID           NO: 25).                                                                      __________________________________________________________________________

[tTF] linker [tTF] DNA was used as the template. The remaining PCRconditions were the same as described in the tTF section. The vectorconstructs and protein purification were again performed as described inthe purification of [tTF]cys section.

D. Chemically Conjugated Dimers

[tTF] Cys monomer, which had been treated with Ellman's reagent toconvert the free Cys to an activated disulfide group, was reduced withhalf a molar equivalent of dithiothreitol. This generated free Cysresidues in half of the molecules. The monomers are conjugatedchemically to form [tTF] Cys--Cys [tTF] dimers. This is done by addingan equal molar amount of DTT to the protected [tTF] Cys at roomtemperature for 1 hr to deprotect and expose the cysteine at theC-terminus of [tTF] Cys. An equal molar amount of protected [tTF] Cys isadded to the DTT/[tTF] Cys mixture and the incubation is continued for18 h at room temperature. The dimers are purified on a G-75 gelfiltration column. Dimers of H₆ -tTF₂₂₀ -cys-C', H₆ -tTF₂₂₁ -cys-C' andH₆ -N'-cys-tTF₂₁₉ were prepared likewise. The Cys [tTF] monomer isconjugated chemically to form dimers using the same method.

EXAMPLE III Synthesis of Tissue Factor Mutants

Three tTF mutants are described that lack the capacity to converttTF-bound Factor VII to Factor VIIa. There is 300-fold less Factor VIIain the plasma compared with Factor VII (Morrissey et al., 1993).Therefore, circulating mutant tTF should be less able to initiatecoagulation and hence exhibit very low toxicity. However, once themutant tTF has localized to the tumor site, as is surprisinglydemonstrated herein, Factor VIIa may be injected to exchange with thetTF-bound Factor VII. The mutated proteins have the sequences shown inSEQ ID NO:8 and SEQ ID NO:9 and are active in the presence of FactorVIIa.

A. [tTF]G164A

The "[tTF]G164A" has the mutant protein structure with the amino acid164 (Gly) of tTF₂₁₉ being replaced by Ala. The Chameleon double-strandedsite directed mutagenesis kit (Stratagene) was used for generating themutant. The DNA template is Gly[tTF] DNA and the sequence of themutagenizing primer is:

    5' CAA GTT CAG CCA AGA AAAC                                (SEQ ID NO:26)

The G164A mutant is represented by SEQ ID NO:9. The vector constructsand protein purification procedures described above were used in thepurification of Gly[tTF].

B. [tTF]W158R

The tryptophan at amino acid 158 of tTF₂₁₉ was mutated to an arginine byPCR™ with a primer encoding this change. Expression, refolding andpurification was as for tTF₂₁₉. The mutated protein has the sequencesshown in SEQ ID NO:8.

C. [tTF]W158R S162A

The [tTF]W158R S162A is a double mutant in which amino acid 158 (Trp) oftTF₂₁₉ is replaced by Arg and amino acid 162 (Ser) is replaced by Ala.The same mutagenizing method is used as described for [tTF] G164A and[tTF]W158R. The mutagenizing primer is:

    5' ACA CTT TAT TAT CGG AAA TCT TCA GCT TCA GGA AAG         (SEQ ID NO:27)

The foregoing vector constructs and protein purification procedures arethe same as used for purifying Gly[tTF].

EXAMPLE IV Preparation of tTF-Bispecific Antibody Adducts and Synthesisof Tissue Factor Conjugates

A. Preparation of tTF-Bispecific Antibody Adducts

Bispecific antibodies were constructed that had one Fab' arm of the10H10 antibody that is specific for a non-inhibitory epitope on tTFlinked to one Fab' arm of antibodies (OX7, Mac51, CAMPATH-2) ofirrelevant specificity. When mixed with tTF, the bispecific antibodybinds the tTF via the 10H10 arm, forming a non-covalent adduct. Thebispecific antibodies were synthesized according to the method ofBrennan et al. (1985;incorporated herein by reference) with minormodifications.

In brief, F(ab')₂ fragments were obtained from the IgG antibodies bydigestion with pepsin (type A; EC 3.4.23.1) and were purified tohomogeneity by chromatography on Sephadcx G100. F(ab')₂ fragments werereduced for 16 h at 20° C. with 5 mM 2-mercaptoethanol in 0.1 M sodiumphosphate buffer, pH 6.8, containing 1 mM EDTA (PBSE buffer) and 9 mMNaAsO₂. Ellman's reagent (ER) was added to give a final concentration of25 mM and, after 3 h at 20° C., the Ellman's derivatized Fab' fragments(Fab'-ER) were separated from unreacted ER on columns of Sephadex G25 inPBSE.

To form the bispecific antibody, Fab'-ER derived from one antibody wasconcentrated to approximately 2.5 mg/ml in an Amicon ultrafiltrationcell and was reduced with 10 mM 2-mercaptoethanol for 1 h at 20° C. Theresulting Fab'-SH was filtered through a column of Sephadex G25 in PBSEand was mixed with a 1:1-fold molar excess of Fab'-ER prepared from thesecond antibody. The mixtures were concentrated by ultrafiltration toapproximately 3 mg/ml and were stirred for 16 h at 20° C. The productsof the reaction were fractionated on columns of Sephadex G100 in PBS.The fractions containing the bispecific antibody (110 kDa) wereconcentrated to 1 mg/ml, and stored at 4° C. in 0.02% sodium azide.

To form the tTF-bispecific antibody adducts, the bispecific antibody wasmixed with a molar equivalent of tTF or derivatives thereof for 1 hourat 4° C. The adduct eluted with a molecular weight of approximately 130kDa on gel filtration columns, corresponding to one molecule ofbispecific antibody linked to one molecule of tTF.

1. Preparation of IgG-H₆ -N'-cys-tTF₂₁₉ and IgG-H₆ -tTF₂₁₉ -cys-C'

To 26 mg IgG at a concentration of 10 mg/ml in N₂ -flushedphosphate-saline buffer was added 250 μg SMPT (Pharmacia) in 0.1 ml dryDMF. After stirring for 30 minutes at room temperature, the solution wasapplied to a column (1.6 cm diameter×30 cm) of Sephadex G25(F)equilibrated in the same buffer. The derivatized IgG was collected in avolume of 10 to 12 ml and concentrated to about 3.5 ml byultrafiltration (Amicon, YM2 membrane). The H₆ -N'-cys-tTF₂₁₉ or H₆-tTF₂₁₉ -cys-C' (15 mg) was reduced by incubation at room temperature inthe presence of 0.2 mM DTT until all Ellman's agent was released (i.e.OD at 412 nm reached a maximum). It was then applied to the SephadexG25(F) column (1.6 cm diameter×30 cm) equilibrated with N₂ -flushedbuffer.

The Cys-tTF (˜15 ml) was added directly to the derivatized IgG solution.The mixture was concentrated to about 5 ml by ultrafiltration andincubated at room temperature for 18 hours before resolution by gelfiltration chromatography on Sephacryl S200. The peak containingmaterial having a molecular weight of 175,000-200,000 was collected.This component consisted of one molecule of IgG linked to one or twomolecules of tTF. The conjugates have the structure: ##STR1##

2. Preparation of Fab'-H6-N'-cys-tTF219

Fab' fragments were produced by reduction of F(ab')₂ fragments of IgGwith 10 mM mercaptoethylamine. The resulting Fab' fragments wereseparated from reducing agent by gel filtration on Sephadex G25. Thefreshly-reduced Fab' fragment and the Ellman's modified H₆-N'-cys-tTF₂₁₉ were mixed in equimolar amounts at a concentration of 20μM. The progress of the coupling reaction was followed by the increasein absorbance at 412 nm due to the 3-carboxylato-4-nitrothiophenolateanion released as a result of conjugation. The conjugate has thestructure:

    Fab'-SS-tTF

B. Synthesis of Tissue Factor Conjugates

1. Chemical Derivatization and Antibody Conjugation

Antibody tTF conjugates were synthesized by the linkage of chemicallyderivatized antibody to chemically derivatized tTF via a disulfide bond.

Antibody was reacted with a 5-fold molar excess of succinimidyloxycarbonyl-α-methyl α-(2-pyridyldithio)toluene (SMPT) for 1 hour atroom temperature to yield a derivatized antibody with an average of 2pyridyldisulphide groups per antibody molecule. Derivatized antibody waspurified by gel permeation chromatography.

A 2.5-fold molar excess of tTF over antibody was reacted with a 45-foldmolar excess of 2-iminothiolane (2IT) for 1 hour at room temperature toyield tTF with an average of 1.5 sulfhydryl groups per tTF molecule.Derivatized tTF was also purified by gel permeation chromatography andimmediately mixed with the derivatized antibody.

The mixture was left to react for 72 hours at room temperature and thenapplied to a Sephacryl S-300 column to separate the antibody-tTFconjugate from free tTF and released pyridine-2-thione. The conjugatewas separated from free antibody by affinity chromatography on aanti-tTF column. The predominant molecular species of the finalconjugate product was the singly substituted antibody-tTF conjugate (Mrapprox. 176,000) with lesser amounts of multiply substituted conjugates(Mr≧approx. 202,000) as assessed by SDS-PAGE.

2. Conjugation of Cysteine-Modified tTF to Derivatized Antibody

Antibody-C[TF] and [tTF]C conjugates were synthesized by direct couplingof cysteine-modified tTF to chemically derivatized antibody via adisulfide bond.

Antibody was reacted with a 12-fold molar excess of 2IT for 1 hour atroom temperature to yield derivatized antibody with an average of 1.5sulfhydryl groups per antibody molecule. Derivatized antibody waspurified by gel permeation chromatography and immediately mixed with a2-fold molar excess of cysteine-modified tTF. The mixture was left toreact for 24 hours at room temperature and then the conjugate waspurified by gel permeation and affinity chromatography as describedabove.

The predominant molecular species of the final conjugate was the singlysubstituted conjugate (Mr approx. 176,000) with lesser amounts ofmultiple substituted conjugates (Mr≧approx. 202,000) as assessed bySDS-PAGE.

3. Conjugation of Cysteine-Modified tTF to Fab' Fragments

Antibody Fab'-C[tTF] and [tTF]C conjugates are prepared. Such conjugatesmay be more potent in vivo because they should remain on the cellsurface for longer than bivalent conjugates due to their limitedinternalization capacity. Fab' fragments are mixed with a 2-fold molarexcess of cysteine-modified tTF for 24 hours and then the conjugatepurified by gel permeation and affinity chromatography as describedabove.

EXAMPLE V Tumor Infraction by Tissue Factor

A. Methods

1. In Vitro Coagulation Assay

This assay was used to verify that tTF, various derivatives and mutantsthereof, and immunoglobulin-tTF conjugates acquire coagulation inducingactivity once localized at a cell surface. A20 lymphoma cells (I-A^(d)positive) (2×10⁶ cells/ml, 50 μl) were incubated for 1 h at roomtemperature with a bispecific antibody (50 μg/ml, 25 μl) consisting of aFab' arm of the B21-2 antibody directed against I-A^(d) linked to a Fab'arm of the 10H10 antibody directed against a non-inhibitory epitope ontTF. The cells were washed at room temperature and varyingconcentrations of tTF, derivatives or mutants thereof, orimmunoglobulin-tTF conjugates were added for 1 hour at room temperature.The bispecific antibody captures the tTF or tTF linked toimmunoglobulin, bringing it into close approximation to the cellsurface, where coagulation can proceed.

The cells were washed again at room temperature, resuspended in 75 μl ofPBS and warmned to 37° C. Calcium (12.5 mM) and citrated mouse or humanplasma (30 μl) were added. The time for the first fibrin strands to formwas recorded. Clotting time was plotted against tTF concentration andcurves compared with standard curves prepared using standard tTF₂₁₉preparations.

In some studies, varying concentrations of recombinant human Factor VIIawere added together with tTF₂₁₉ and mutants thereof, to determinewhether coagulation rate was enhanced by the presence of Factor VIIa.

2. Factor Xa Production Assays

This assay is useful in addition to or as an alternative to the in vitrocoagulation assay to demonstrate that tTF and immunoglobulin-tTFconjugates acquire coagulation inducing activity once localized at acell surface. The assay measures factor X to Xa conversion rate by meansof a chromophore-generating substrate (S-2765) for factor Xa.

A20 cells (2×10⁷ cells) were suspended in 10 ml medium containing 0.2%w/v sodium azide. To 2.5 ml cell suspension were added 6.8 μg ofB21-2/10H10 "capture" bispecific antibody for 50 minutes at roomtemperature. The cells were washed and resuspended in 2.5 ml mediumcontaining 0.2% w/v sodium azide. The tTF and immunoglobulin-tTFconjugates dissolved in the same medium were distributed in 100 μlvolumes at a range of concentrations into wells of 96-well microtiterplates. To the wells was then added 100 μl of the cell/bispecificantibody suspension. The plates were incubated for 50 minutes at roomtemperature.

The plates were centrifuged, the supernatants were discarded and thecell pellets were resuspended in 250 μl of Wash Buffer (150 mM NaCl;50mM Tris-HCl, pH 8; 0.2% w/v bovine serum albumin). The cells were washedagain and cells resuspended in 100 μl of a 12.5-fold dilution of ProplexT (Baxter, Inc.) containing Factors II, VII, IX and X in Dilution Buffer(Wash Buffer supplemented with 12.5 mM calcium chloride). Plates wereincubated at 37° C. for 30 minutes. To each well was added Stop Solution(12.5 mM sodium ethylenediaminetetracetic acid (EDTA)) in wash buffer.Plates were centrifuged. 100 μl of supernatant from each well were addedto 11 μl of S-2765(N-α-benzyloxycarbonyl-D-Arg-L-Gly-L-Arg-p-nitroanilide dihydrochloride,Chromogenix AB, Sweden). The optical density of each solution wasmeasured at 409 nm. Results were compared to standard curves generatedfrom standard tTF₂₁₉.

3. In Vivo Tumor Thrombosis

This model was used to demonstrate that tTF and immunoglobulin-tTFconjugates induced thrombosis of tumor blood vessels and caused tumorinfarction in vivo.

Tumor test systems were of four types: i) 3LL mouse lung carcinomagrowing subcutaneously in C57BL/6 mice; ii) C1300 mouse neuroblastomagrowing subcutaneously in BALB/c nu/nu mice; iii) HT29 human colorectalcarcinoma growing subcutaneously in BALB/c nu/nu mice; and iv) C1300 Muγmouse neuroblastoma growing subcutaneously in BALB/c nu/nu mice. TheC1300 Muγ tumor is an interferon-y secreting transfectant derived fromthe C1300 tumor (Watanabe et al., 1989).

Further, the C1300 (Muγ) tumor model of (Burrows, et al.,1992;incorporated herein by reference) was employed and modified asfollows: (i) antibody B21-2 was used to target I-A^(d) ; (ii) C1300(Muγ)tumor cells, a subline of C1300(Muγ)12 tumor cells, that grewcontinuously in BALB/c nu/nu mice were used; and (iii) tetracycline wasomitted from the mice's drinking water to prevent gut bacteria frominducing I-A^(d) on the gastrointestinal epithelium. Unlikeimmunotoxins, coaguligands and Tissue Factor constructs do not damageI-A^(d) -expressing intestinal epithelium.

4. Tumor Establishment

To establish tumors, 10⁶ to 1.5×10⁷ tumor cells were injectedsubcutaneously into the right anterior flank of the mice. When tumorshad grown to various sizes, mice were randomly assigned to differentstudy groups. Mice then received an intravenous injection of 0.5 mg/kgof tTF alone or linked to IgG, Fab', or bispecific antibody. Other micereceived equivalent quantities IgG, Fab' or bispecific antibody alone.The injections were performed slowly into one of the tail veins overapproximately 45 seconds, usually followed by 200 μl of saline.

In some studies, the effect of administering cancer chemotherapeuticdrugs on the thrombotic action of tTF on tumor blood vessels wasinvestigated. Mice bearing subcutaneous HT29 human colorectal tumors of1.0 cm diameter were given intraperitoneal injections of doxorubicin (1mg/kg/day), camptothecin (1 mg/kg/day), etoposide (20 mg/kg/day) orinterferon gamma (2×10⁵ units/kg/day) for two days before the tTFinjection and again on the day of the tTF injection.

Twenty-four hours after being injected with tTF or immunoglobulin-tTFconjugates, the mice were anesthetized with metophane and wereexsanguinated by perfusion with heparinized saline. Tumors and normaltissues were excised and immediately fixed in 3% (v/v) formalin.Paraffin sections were cut and stained with hematoxylin and eosin. Bloodvessels having open lumens containing erythrocytes and blood vesselscontaining thrombi were counted. Paraffin sections were cut and stainedwith hematoxylin and eosin or with Martius Scarlet Blue (MSB) trichromefor the detection of fibrin.

5. Anti-Tumor Effects

Accepted animal models were used to determine whether administration oftTF or immunoglobulin-tTF conjugates suppressed the growth of solidtumors in mice. The tumor test systems were: i) L540 human Hodgkin'sdisease tumors growing in SCID mice; ii) C1300 Muγ(interferon-secreting) neuroblastoma growing in nu/nu mice; iii) H460human non-small cell lung carcinoma growing in nu/nu mice. To establishsolid tumors, 1.5×10⁷ tumor cells were injected subcutaneously into theright anterior flank of SCID or BALB/c nu/nu mice (Charles River Labs.,Wilmingham, Mass.). When the tumors had grown to various diameters, micewere assigned to different experimental groups, each containing 4 to 9mice.

Mice then received an intravenous injection of 0.5 mg/kg of tTF alone orlinked to bispecific antibody. Other mice received equivalent quantitiesof bispecific antibody alone. The injections were performed over ˜45seconds into one of the tail veins, followed by 200 μl of saline. Theinfusions were repeated six days later. Perpendicular tumor diameterswere measured at regular intervals and tumor volumes were calculated.

B. Results

1. In vitro Coagulation by tTF and Variants

To target tTF to I-A^(d) on tumor vascular endothelium, the inventorsprepared a bispecific antibody with the Fab' arm of the B21-2 antibody,specific for I-A^(d), linked to the Fab' arm of the 10H10 antibody,specific for a non-inhibitory epitope on the C-module of tTF. Thisbispecific antibody, B21-2/10H10, mediated the binding of tTF in anantigen-specific manner to I-A^(d) on A20 mouse B-lymphoma cells invitro. When mouse plasma was added to A20 cells to which tTF had beenbound by B21-2/10H10, it coagulated rapidly. Fibrin strands were visible36 seconds after the addition of plasma to antibody-treated cells, ascompared with 164 seconds when plasma was added to untreated cells (FIG.4A). Only when tTF was bound to the cells was this enhanced coagulationobserved: no effect on coagulation time was seen with cells incubatedwith tTF alone, with homodimeric F(ab')₂, with Fab' fragments, or withtTF plus bispecific antibodies that had only one of the twospecificities needed for binding tTF to A20 cells.

tTF₂₁₉ prepared as in Example I had identical ability to a "standard"tTF₂₁₉ preparation obtained from Dr. Thomas Edgington (The ScrippsResearch Institute, La Jolla, Calif.) to induce coagulation of mouse orhuman plasma after its binding via B21-2/10H10 bispecific antibody toA20 lymphoma cells (FIG. 5). Mouse plasma coagulated in 50 seconds whenboth the preparation of tTF₂₁₉ of Example I and the "standard" tTF wereapplied to the cells at 3×10⁻⁹ M. Thus, the tTF₂₁₉ prepared as describedherein appears to be correctly refolded and fully active.

There was a linear relationship between the logarithm of the number oftTF molecules bound to the cells and the rate of plasma coagulation bythe cells (FIG. 4B). In the presence of cells alone, plasma coagulatedin 190 seconds, whereas at 300,000 molecules of tTF per cell coagulationtime was 40 seconds. Even with only 20,000 molecules per cell,coagulation was faster (140 seconds) than with untreated cells. These invitro studies showed that the thrombogenic potency of tTF is enhanced bycell surface proximity mediated through antibody-directed binding toClass II antigens on the cell surface.

H₆ -N'-cys-tTF₂₁₉ and H₆ -tTF₂₁₉ -cys-C' were as active as tTF atinducing coagulation of plasma once bound via the bispecific antibody toA20 cells. Plasma coagulated in 50 seconds when H₆ -N'-cys-tTF₂₁₉ and H₆-tTF₂₁₉ -cys-C' were applied at 3×10⁻⁹ M, the same concentration as fortTF (FIG. 5). Thus, mutation of tTF to introduce a (His)₆ sequence and aCys residue at the N' or C' terminus does not reduce itscoagulation-inducing activity.

H₆ -tTF₂₂₀ -cys-C', tTF₂₂₀ -cys-C', H₆ -tTF₂₂₁ -cys-C' and tTF₂₂₁-cys-C' were as active as tTF₂₁₉ at inducing coagulation of plasma oncelocalized on the surface of A20 cells via the bispecific antibody,B21-2/10H10. With all samples at 5×10⁻¹⁰ M, plasma coagulated in 50seconds (FIG. 6 and FIG. 7).

2. In Vitro Coagulation by tTF Dimers

H₆ -N'cys-tTF₂₁₉ dimer was as active as tTF₂₁₉ itself at inducingcoagulation of plasma once localized on the surface of A20 cells via thebispecific antibody, B21-2/10H10. At a concentration of 1-2×10⁻¹⁰ M,both samples induced coagulation in 50 seconds (FIG. 8). In contrast, H₆-tTF₂₂₁ -cys-C' dimer was 4-fold less active than H₆ -tTF₂₂₁ -cys-C'monomer or tTF₂₁₉ itself. At a concentration of 4×10⁻⁹ M, H₆ -tTF₂₂₁-cys-C' dimer induced coagulation of plasma in 50 seconds, whereas thecorresponding monomer needed to be applied at 1×10⁻⁹ M for the sameeffect on coagulation.

3. In vivo Tumor Thrombosis

A histological study was performed to determine whether intravenousadministration of the B21-2/10H10-tTF coaguligand induced selectivethrombosis of tumor vasculature in mice bearing subcutaneous C1300(Muγ)neuroblastomas of 0.8 to 1.0 cm diameter (FIG. 9). Within 30 minutes,all vessels throughout the tumor were thrombosed, containing occlusiveplatelet aggregates, packed erythrocytes, and fibrin. At this time,tumor cells were indistinguishable histologically from tumor cells ofuntreated mice.

After 4 hours, however, there were signs of tumor cell injury. Themajority of tumor cells had separated from one another and had pyknoticnuclei, and the tumor interstitium commonly contained erythrocytes. By24 hours, the tumor showed advanced necrosis, and by 72 hours, theentire central region of the tumor had condensed into amorphous debris.These studies indicated that the predominant occlusive effect of theB21-2/10H10-tTF coaguligand on tumor vessels is mediated through bindingto Class II antigens on tumor vascular endothelium.

Surprisingly it was observed that there was a non-specific thromboticaction of tTF discernible in tumor vessels at later times: In tumorsfrom mice which had been injected 24 hours previously with tTF alone ortTF mixed with the control bispecific antibody, OX7/10H10, the tumorsassumed a blackened, bruised appearance starting within 30 minutes andbecoming progressively more marked up to 24 hours. A histological studyrevealed that 24 hours after injection of tTF₂₁₉ practically all vesselsin all regions of the tumor were thrombosed (FIG. 9). Vessels containedplatelet aggregates, packed red cells and fibrin. The majority of tumorcells had separated from one another and had developed pyknotic nucleiand many regions of the tumors were necrotic. These were most pronouncedin the tumor core. Erythrocytes were commonly observed in the tumorinterstitium.

It is possible that the resident thrombogenic activity of tumorvasculature (Zacharski, et al., 1993) renders these vessels moresusceptible to thrombosis even by untargeted tTF. Alternatively,enhanced procoagulant changes might have been induced by thetumor-derived IFNγ.

Similar results were obtained when tTF₂₁₉ was administered to micebearing large C1300 tumors (>1000 mm³). Again, virtually all vesselswere thrombosed 24 hours after injection (FIG. 10). Thus, the effectsobserved on C1300 Muγ tumors were not related to the interferon γsecretion by the tumor cells.

Further studies were performed in C57BL/6 mice bearing large (>800 mm³)3LL tumors. Again, thrombosis of tumor vessels was observed, thoughsomewhat less pronounced than with the C1300 and C1300 Muγ tumor. Onaverage 62% of 3LL tumor vessels were thrombosed (FIG. 11).

Vessels in small (<500 mm³) C1300 and C1300 Muγ were largely unaffectedby tTF₂₁₉ administration. Thus, as the tumors grow, their susceptibilityto thrombosis by tTF₂₁₉ increases. This is possibly because cytokinesreleased by tumor cells or by host cells that infiltrate the tumoractivate the tumor vascular endothelium, inducing procoagulant changesin the vessels.

Coaguligand treatment was well tolerated, mice lost no weight andretained normal appearance and activity levels. At the treatment dose of0.6 mg/kg B21-2/10H10 plus 0.5 mg/kg tTF, toxicity was observed in onlytwo of forty mice (thrombosis of tail vein). It is important to notethat neither thrombi, nor histological or morphological abnormalitieswere visible in paraffin sections of liver, kidney, lung, intestine,heart, brain, adrenals, pancreas, or spleen from the tumor-bearing mice30 minutes or 24 hours after administration of coaguligand or free tTF.Furthermore, no signs of toxicity (behavioral changes, physical signs,weight changes) were observed in treated animals.

4. Anti-Tumor Effects

The inventors next investigated whether intravenous administration ofthe B21-2/10H10-tTF coaguligand could inhibit the growth of large (0.8to 1.0 cm diameter) tumors in mice. The pooled results from threeseparate studies indicate that mice receiving B21-2/10H10-tTFcoaguligand had complete tumor regressions lasting four months or more.These anti-tumor effects were significantly greater than for all othertreatment groups (FIG. 12A).

Surprisingly, the inventors found that the anti-tumor effect of theB21-2/10H10-tTF coaguligand was attributable, in part, to a non-targetedeffect of tTF. Tumors in mice receiving tTF alone or mixed with controlbispecific antibodies (CAMPATH II/10H10 or B21-2/OX7) grew significantlymore slowly than tumors in mice receiving antibodies or saline alone(FIG. 12A; FIG. 12B).

Mice bearing small (300 mm³) C1300 Muγ tumors were injectedintravenously with 16-20 μg tTF₂₁₉. The treatment was repeated onc weeklater. The first treatment with tTF₂₁₉ had a slight inhibitory effect ontumor growth, consistent with the lack of marked thrombosis observedwith small tumors above (FIG. 12B). The second treatment had asubstantially greater, statistically significant (P<0.01), effect ontumor growth, probably because the tumors had increased in size. Oneweek after the second treatment with tTF₂₁₉, tumors were 60% of the sizeof tumors in mice receiving diluent alone. The greater effectiveness ofthe second injection probably derives from the greater thrombotic actionof tTF₂₁₉ on vessels in large tumors, observed above.

Similar anti-tumor effects were observed in mice bearing H460 human lungcarcinomas (FIG. 13). The first treatment with tTF₂₁₉ was given when thetumors were small (250 mm³) and had little effect on growth rate. Thesecond treatment with tTF₂₁₉ was given when the tumors were larger (900mm³) and caused the tumors to regress to 550 mm³ before regrowing.

Anti-tumor effects were also observed in mice bearing HT29 humancolorectal carcinomas (FIG. 14). Nu/nu mice bearing large (1200 mm³)tumors on their flanks were injected intravenously with tTF₂₁₉ or PBS(control), and growth of the tumors was monitored each day for 10 days.The tumors in the tTF₂₁₉ treated mice discontinued growth for about 7days after treatment, whereas the tumors in mice treated with PBScontinued to grow unchecked.

In animals that did not show complete tumor regression afterB21-2/10H10-tTF coaguligand treatment, the tumors grew back from asurviving microscopic rim of cells at the periphery of the tumor.Immunohistochemical examination of these tumors revealed that thevascular endothelium at the invading edge of the tumors lackeddetectable Class II antigens, consistent with a lack of thrombosis ofthese vessels by the coaguligand permitting local tumor cell survival.Thus, coadministration of a drug acting on the tumor cells themselveswould likely improve efficacy, as has been observed with anotherantivascular therapy (Burrows and Thorpe, 1992;Burrows and Thorpe1993;Burrows and Thorpe 1994;U.S. Ser. Nos. 07/846,349; 08/205,330;08/295,868;and 08/350,212).

The inventors previously demonstrated that a powerfully cytotoxic ricinA-chain immunotoxin directed against the tumor cells themselves wasvirtually devoid of anti-tumor activity when administered to mice withlarge C1300(Muγ) tumors (Burrows and Thorpe, 1993; U.S. Ser. Nos.07/846,349; 08/205,330; 08/295,868;and 08/350,212). The lack of activitywas due to the inability of the immunotoxin to gain access to tumorcells in large tumor masses, thus attesting to the comparativeeffectiveness of coaguligand therapy.

The studies using coaguligands confirm the therapeutic potential ofselective initiation of the blood coagulation cascade in tumorvasculature (U.S. Ser. Nos. 08/273,567; 08/482,369; 08/485,482;08/487,427; 08/479,733; 08/472,631; 08/479,727;and 08/481,904). Theinduction of tumor infarction by targeting a thrombogen to tumorendothelial cell markers is therefore an effective anti-cancer strategyand may even result in the eradication of primary solid tumors andvascularized metastases.

The successful use of tTF alone or tTF immunoconjugates with an antibodyof irrelevant specificity was initially a surprising outcome of thetargeting studies. Although mice receiving tTF alone did not havecomplete tumor regressions, it is clear that the surprising anti-tumoractivity of tTF renders this and functionally related TF derivativesuseful in the treatment of solid tumors. The benefits of suchcompositions as detailed herein are far reaching and include the lack ofside effects from the use of such TFs. Further, it is well within theskill of those in the art to produce the type of tTF compositionspresented in the instant invention. Such compositions can then beemployed in the treatment of solid tumors alone or in combination withother anti-cancer agents.

EXAMPLE VI Coagulation of Mouse Plasma by Immunogloubulin-TF Conjugates

IgG-H₆ -N'-cys-tTF₂₁₉ was active at inducing coagulation of mouse plasmawhen localized on the surface of A20 cells by means of the bispecificantibody, B21-2/10H10. It induced coagulation in 50 seconds when appliedat a tTF concentration of 5×10⁻⁹ M as compared with 1×10⁻⁹ M fornon-conjugated tTF₂₁₉ and H₆ -N'-cys-tTF₂₁₉ (FIG. 15). The coagulationinducing activity of IgG-H₆ -N'-cys-tTF₂₁₉ is therefore reduced 5-foldrelative to unconjugated H₆ -N'-cys-tTF₂₁₉ or tTF₂₁₉ itself.

The slight reduction upon IgG conjugation could be because the IgGmoiety of IgG-H₆ -N'-cys-tTF₂₁₉ impedes access of the B21-2/10H10bispecific antibody to the tTF moiety (i.e., an artifactual reductionrelated to the assay method). It is probably not because the IgG moietyof IgG-H6-N'-cys-tTF219 interferes with formation of the coagulationinitiation complexes because, in prior work, the inventors have foundthat the tTF moiety in an analogous construct, B21-2 IgG-H₆-N'-cys-tTF₂₁₉, is as active as tTF bound via B21-2/10H10 to I-A^(d)antigens on A20 cells (FIG. 16). Similarly, B21-2 IgG-H₆ -tTF₂₁₉ -cys-C'was as active at inducing coagulation as was the N'-linked conjugation(FIG. 16).

IgG-H₆ -N'-cys-tTF₂₁₉ and Fab'-H₆ -N'-cys-tTF₂₁₉ were tested for theirability to convert Factor X to Xa in the presence of Factors II, VII andIX, once localized on the surface of A20 lymphoma cells by means of thebispecific antibody, B21-2/10H10. The Fab'-tTF construct was as activeas H₆ -N'-cys-tTF₂₁₉ itself at inducing Xa formation. The IgG-tTFconstruct was slightly (2-fold) less active than H₆ -N'-cys-tTF₂₁₉itself (FIG. 17).

EXAMPLE VII Inhibition of Growth of C1300 Muγ Tumors byImmunpglobulin-TF Conjugate

Mice bearing small (300 mm³) subcutaneous C1300 Muγ tumors were treatedwith tTF₂₁₉ or with a complex of tTF₂₁₉ and a bispecific antibody, OX7Fab'/10H10 Fab', not directed to a component of the tumor environment.The treatment was repeated 6 days later (FIG. 18). The bispecificantibody was simply designed to increase the mass of the tTF₂₁₉ from 25kDa to 135 kDa, and thus prolong its circulatory half life, and was notintended to impart a targeting function to tTF.

Tumors in mice treated with the immunoglobulin-tTF conjugate grew moreslowly than those in mice receiving tTF₂₁₉ alone. Fourteen days afterthe first injection, tumors were 55% of the size of those in controlsreceiving diluent alone. In mice receiving tTF₂₁₉ alone, tumors were 75%of the size in controls receiving diluent alone (FIG. 18).

EXAMPLE VIII Enhancement of Anti-Tumor Activity of Immunoglobulin-tTFConjugate by Etoposide

Mice bearing L540 human Hodgkin's disease tumors were treated with acomplex of tTF₂₁₉ and a bispecific antibody together with theconventional anti-cancer drug, etoposide. Etoposide greatly enhanced theaction of the immunoglobulin-tTF conjugate. In this tumor model alone,mice receiving the antibody-tTF complex alone showed little reduction intumor growth relative to tumors in mice receiving diluent alone (FIG.19).

In contrast, tumors in mice receiving both etoposide and theimmunoglobulin-tTF conjugate regressed in size and did not recommencegrowth for seventeen days. At the end of the study (day 20), tumors inmice receiving etoposide plus immunoglobulin-tTF were an average of 900mm³ in volume as compared with 2300 mm³ in mice treated with diluent and2000 mm³ in mice treated with immunoglobulin-tTF alone. In micereceiving etoposide alone, tumors averaged 1400 mm³ on day 14 (FIG. 19).These results indicate that etoposide may predispose tumor vessels tothrombosis by tTF or immunoglobulin-tTF conjugates. Irrespective of themechanism, the results clearly show advantageous combination of TF, or aTF-conjugate, with a classical chemotherapeutic agent.

EXAMPLE IX Enhancement of Plasma Coagulation by VIIa

The ability of cell-associated tTF₂₁₉ to induce coagulation of mouse orhuman plasma was strongly enhanced in the presence of free Factor VIIa(FIG. 20). In the absence of Factor VIIa, A20 cells treated withB21-2/10H10 bispecific antibody and 10⁻¹⁰ M tTF₂₁₉ coagulated plasma in60 seconds, whereas in the presence of 13.5 nM Factor VIIa, itcoagulated plasma in 20 seconds (FIG. 20). This represents approximatelya 100-fold enhancement in the coagulation-inducing potency of tTF in thepresence of Factor VIIa. Even in the presence of 0.1 nM Factor VIIa, a2-5 fold increase in coagulation-inducing potency of tTF was observed.

This finding leads to the aspects of the invention that concern thecoadministration of Factor VIIa along with tTF or derivatives thereof,or with immunoglobulin-tTF conjugates, in order to enhance tumor vesselthrombosis in vivo.

EXAMPLE X Reduced Coagulation of Mouse Plasma by TF Factor VIIActivation Mutants

Mutations in W158 and G164 of tTF₂₁₉ have been reported to reducemarkedly the ability of TF to induce coagulation of recalcified plasma(Ruf et al., 1992;Martin et al, 1995). Residues 157-167 of TF appear tobe important in accelerating activation of Factor VII to Factor VIIa,but not the binding of Factor VII to TF. The inventors mutated W158 to Rand G164 to A and determined whether the mutants acquired the ability tocoagulate plasma once localized by means of a bispecific antibody,B21/2-10H10, on the surface of A20 cells. It was found that the mutantswere 30-50-fold less effective than was tTF₂₁₉ at inducing coagulationof plasma (FIG. 21).

EXAMPLE XI Restoration of Coagulating Ability of Factor VII ActivationMuntants by Factor VIIa

Mutant tTF₂₁₉ (G164A) is a very weakly coagulating mutant of tTF₂₁₉(Ruf, et al, 1992). The mutation is present in a region of TF (aminoacids 157-167) thought to be important for the conversion of Factor VIIto Factor VIIa. Thus, addition of Factor VIIa to cells coated withbispecific antibody and tTF₂₁₉ (G164A) would be reasoned to induce thecoagulation of plasma. In support of this, A20 cells coated withB21-2/10H10 followed by tTF₂₁₉ (G164A) had increased ability to inducecoagulation of plasma in the presence of Factor VIIa (FIG. 22). Additionof Factor VIIa at 1 nM or greater produced only marginally slowercoagulation times than observed with tTF₂₁₉ and Factor VIIa at the sameconcentrations.

Mutant tTF₂₁₉ (W158R) gave similar results to tTF₂₁₉ (G164A). Again,addition of Factor VIIa at 1 nM or greater to A20 cells coated withB21-2/10H10 followed by tTF₂₁₉ gave only marginally slower coagulationtimes than did tTF₂₁₉ and Factor VIIa at the same concentrations.

These results support those aspects of the invention that provide thattTF₂₁₉ (G164A) or tTF₂₁₉ (W158R), when coadministered with Factor VIIato tumor-bearing animals, will induce the thrombosis of tumor vessels.This approach is envisioned to be advantageous because tTF (G164A), tTF(W158R) or Factor VIIa given separately are practically non-toxic tomice, and the same is reasonably expected in humans. Coadministration ofthe mutant tTF and Factor VIIa is expected not to cause toxicity, yet tocause efficient thrombosis of tumor vessels. Giving mutant tTF togetherwith Factor VIIa is thus contemplated to result in an improvedtherapeutic index relative to tTF₂₁₉ plus Factor VIIa.

EXAMPLE XII Enhanced Anti-Tumor Activity of Activation Mutants andFactor VIIa

For these studies, the inventors chose the HT29 (human colorectalcarcinoma) xenograft tumor model. HT29 cells (10⁷ cells/mouse) weresubcutaneously injected into BALB/c nu/nu mice. Tumor dimensions weremonitored and animals were treated when the tumor size was between 0.5and 1.0 cm³. Animals were given an intravenous injection of one of thefollowing: tTF₂₁₉ (16 μg), tTF₂₁₉ (16 μg)+Factor VIIa (1 μg), tTF₂₁₉(G164A) (64 μg), tTF₂₁₉ (G164A) (64 μg)+Factor VIIa (1 μg), Factor VIIaalone (1 μg), or saline.

Animals were sacrificed 24 hours after treatment, perfused with salineand heparin and exsanguinated. Tumors and organs were collected,formalin fixed and histological sections were prepared. The average areaof necrosis in sections of the tumors was quantified and calculated as apercentage of the total area of tumor on the section.

In these small HT29 tumors, analysis of tumor sections from animalstreated with saline, Factor VIIa, tTF₂₁₉ or tTF₂₁₉ (G164A) showed somenecrosis (FIG. 23). The tTF-induced tumor necrosis was the mostdeveloped, although this was not as striking, on this occasion, asresults from earlier studies using different tumor models and/or largetumors. An analysis of tumor sections from animals treated with tTF₂₁₉+Factor VIIa or tTF₂₁₉ (G164A)+Factor VIIa revealed considerablenecrosis (12.5% and 17.7% respectively; FIG. 23) and a strongcorrelation between newly thrombosed blood vessels and areas ofnecrosis. The combined use of Factor VIIa with TF, even a TF constructwith particularly deficient in vitro coagulating activity, is thereforea particularly advantageous aspect of the present invention. As the HT29tumor model is difficult to thrombose in general and these tumors weresmall in size, these results are likely to translate to even furtherstriking results in other systems and in humans.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. More specifically, it will beapparent that certain agents which are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988.

Abraham et al., Science, 233 :545-548, 1986.

Abrams and Oldham, Monoclonal Antibody Therapy of Human Cancer, Foon andMorgan (Eds.), Martinus Nijhoff Publishing, Boston, pp. 103-120, 1985.

Ausubel et al., Current Protocols in Molecular Biology, GreenePublishing Associates and Wiley Interscience, N.Y., 1989.

Bach et al., Biochemistry, 25, 4007-4020, 1986.

Bauer, et al., Vox Sang, 61:156-157, 1991.

Baxter, et al., Micro. Res., 41(1):5-23, 1991.

Bevilacqua, et al., Proc. Natl. Acad. Sci. USA, 84:9238-9242, 1987.

Bhagwat et al., Nature, 316:511-513, 1985.

Bicknell and Harris, Seminars in Cancer Biology, 3:399-407, 1992.

Birembaut et al., J. Pathology, 145:283-296, 1985.

Bjomdahl et al., Eur. J. Immunol., 19:881-887, 1989.

Bolhuis et al., J. Immunol., 149:1840-1846, 1992.

Borden et al., Cancer, 65:800-814, 1990.

Brennan et al., Science, 229:81-83, 1985.

Brinkmann et al., Proc. Natl. Acad. Sci., 88(19):8616-8620, 1991.

Broze, Seminars in Hematol., 29:159-169, 1992.

Burchell et al., J. Immunol., 131(1):508-513, 1983.

Burrows et al., Cancer Res., 52:5965-5962, 1992.

Burrows et al., Cancer Res., 51:4768-4775, 1991.

Burrows and Thorpe, Proc. Natl. Acad. Sci., USA, 90:8996-9000, 1993.

Burtin et al., Cancer, 31:719-726, 1983.

Byers and Baldwin Immunol., 65:329-335, 1988.

Campbell, In: Monoclonal Antibody Technology, Laboratory Techniques inBiochemistry and Molecular Biology, Vol. 13, Burden and Von Knippenberg(Eds.), Elseview, Amsterdam, pp. 75-83, 1984.

Chen et al., J. Immunol., 145:8-12, 1990.

Cherwinski et al., J. Exp. Med., 166:1229-1244, 1989.

Clark et al., Biochem. Biophys. ACTA, 867:244-251, 1986.

Clark et al., Int. J. Cancer, 2:15-17, 1988.

Clark et al., Cancer Res., 51:944-948, 199 1.

Colcher et al., Cancer Res., 47:1185 and 4218, 1987.

Collins et al., Proc. Natl. Acad. Sci. USA, 81:4917-4921, 1984.

Cotran et al., J. Exp. Med., 164:661-666, 1986.

Daar el al., Transplantation, 38(3):293-298, 1984.

Davie et al., Biochem., 30: 10363-10310, 1991.

Davies and Wlodawer, FASEB J, 9:50-56, 1995.

Davis and Preston, Analytical Biochemistry, 116(2):402-407, 1981.

DeFranco, Nature, 352:754-755, 1991.

deLeij et al., Bispecific Antibodies and Targeted Cellular Cytotoxicity,Romet-Lemonne et al., p. 249, 1991.

Denekamp et al., Brit. J. Cancer, 461:711-720, 1982.

Dewerchin et al., Blood, 78(4):1005-1018, 1991.

Di Scipio el al., Biochemistry, 16:5253-5260, 1977.

Dillman et al., Antibody Immunocon. Radiopharm., 1:65-77, 1988.

Drake et al., J. Cell Biol., 109:389-95, 1989.

Dustin et al., J. Immunol., 137:245-254, 1986.

Dvorak et al., J. Exp. Med., 174:1275-1278, 1991.

Edgington et al., Thrombosis and Haemostatis, 66(1):67-79, 1991.

Embleton et al., Br. J. Cancer, 63(5):670-674, 1991.

Epenetos el al., Cancer Res., 46:3183-3191, 1986.

Fair, Blood, 62:784-791, 1983.

Fair et al., J. Biol. Chem., 262, 11692-11698, 1987.

Ferrara, J. Cell. Biochem., 47:211-218, 1991.

Flavell et al., Br. J. Cancer, 64(2):274-280, 1991.

Flavell et al., Br. J. Cancer, 65:545-551, 1992.

Folkman, Adv. Cancer Res., 43:175-230, 1985a

Folkman, In: Important Advances in Oncology, Part I, DeVita et al.,(Eds.), J B Lippincott, Philadelphia, pp. 42-62, 1985b.

Fox et al., J. Biol. Resp., 9:499-511, 1990.

Frelinger III, et al., J. Biol. Chem., 265(11):6346-6352, 1990.

Frelinger III, et al., J. Biol. Chem., 266(26):17106-17111, 1991.

French et al., Cancer Res., 51:2358-2361, 1991.

Galfre et al., Methods Enzymol., 73:1-46, 1981.

Gefter et al., Somatic Cell Genet., 3:231-236, 1977.

Geppert et al., Immunological Reviews, 117:5-66, 1990.

Ghose and Blair, CRC Critical Reviews in Therapeutic Drug CarrierSystems, 3:262-359, 1987.

Ghose, CRC Critical Review in Therapeutic Drug Carrier Systems,3:262-359, 1982.

Ghose et al., Meth. Enzymology, 93:280-333, 1983.

Ghose et al., CRC Critical Reviews in Therapeutic Drug Carrier Systems,3:262-359, 1987.

Giles et al., Brit. J. Haematol., 69:491-497, 1988.

Glennie et al., J. Immunol., 139:2367-2375, 1987.

Glennic et al., 1988

Goding, In: Monoclonal Antibodies: Principles and Practice, 2d Ed.,Academic Press, Orlando, Fla., pp. 60-61, 65-66, 71-74, 1986.

Gougos and Letarte, J. Immunol., 141:1925-1933, 1988.

Groenewegen et al., Nature, 316:361-363, 1985.

Hagen, et al., Proc. Natl. Acad. Sci. U.S.A., 83:2412-2416, 1986.

Hailing et al., Nucl. Acids Res., 13:8019-8033, 1985.

Hammerling, Transplant Rev., 30:64-82, 1976.

Hattey et al., Thrombosis Research, 45(5):485-495, 1987.

Hayward et al., Biological Chemistry, 266(11):7114-7120, 1991.

Hess et al., Transplantation, 6:1232-1240, 1991.

Heynen et al., J. Clin. Invest., 94:1098-1112, 1994.

Jain, Cancer Meta. Rev., 9(3):253-266, 1990.

June et al., Mol. Cell Biol., 12:4472-4481, 1987.

June et al., Immunology Today, 11(6):211-216, 1990.

Juweid et al., Cancer Res., 52:5144-5153, 1992.

Kandel et al., Cell, 66:1095-1104, 1991.

Kim et al., Nature, 362:841-844, 1993.

Kimura et al., Immunogenetics, 11:373-381, 1983.

Kisiel, J. Biol. Chem., 254(23):12230-12234, 1979.

Klagsburn and Folkman, Angiogenesis Handbook of ExperimentalPharmacology, Vol. 95, Sporn and Roberts, Springer-Verlag, Berlin, pp.549-586, 1990.

Kohler and Milstein, Nature, 256:495-497, 1975.

Kohler and Milstein, Eur. J. Immunol., 6:511-519, 1976.

Krishnaswamy et al., J. Biol. Chem., 267:26110-26120, 1992.

Kyte and Doolittle, J. Mol. Biol., 157(1):105-132, 1982.

Lamb et al., Eur. J. Biochem., 148:265-270, 1985.

Lee et al., Methods in Enzymology, 237:146-164, 1994.

Leith et al., British J. Cancer, 66(2):345-348, 1992.

Lord et al., In: Genetically Engineered Toxins, Frank (Ed.), M. DekkerPubl., p. 183, 1992.

Lowder et al., Blood, 69:199-210, 1987.

Lowe et al., Immunol. Lett., 12:263-269, 1986.

Maedaet al., J. Invest. Derm., 97:183-189, 1991.

Manabe et al., J. Lab. Clin. Med., 104(3):445-454, 1984.

Martin, FASEB J, 9:852-859, 1995.

Massoglia et al., J. Cell. Phys., 132:531-537, 1987.

Mazzocchi et al., Cancer Immunol. Immuother., 32:13-21, 1990.

Mignatti et al., J. Cell. Biol., 113:1193-1201, 1991.

Miotti et al., Cancer Res., 65:826, 1985.

Moroi and Aoki, J. Biol. Chem., 251(19):5956-5965, 1976.

Morrissey et al., Cell, 50:129-135, 1987.

Morrissey et al., Thrombosis Research, 52:247-261, 1988.

Morrissey et al., Blood, 81:734-744, 1993.

Mueller et al., Proc. Natl. Acad. Sci. USA 89:11832-11836, 1992.

Murray, Clauss, Thurston, Stern, Int. J. Radiat. Biol., 60:273-277,1991.

Nakamura, Prog. Growth Factor Res., 3:67-86, 1991.

Nawroth, Handley, Matsueda, de Waal., Gerlach, Blohm, Stern, J. Exp.Med., 168:637-648, 1988.

Nawroth and Stern, J. Exp. Med., 163:740-745, 1986.

Nelson, Cancer Cells, 3 (5) p163-72, 1991.

Nemerson, Blood, 71(1):1-8, 1988.

Nitta et al., Lancet, 335:368-371, 1990.

Nolan and Kennedy, 1990

O'Brien et al., J. Clin. Invest., 82:206-211, 1988.

O'Connell et al., J. Immunol., 144(2):521-525, 1990.

O'Hare et al., FEBS Lett, 210:731, 1987.

Ogata, J. Biol. Chem., 256:20678-20685, 1990.

Ogawa, Shreeniwas, Brett, Clauss, Furie, Stern, Brit. J. Haematol.,75:517-524, 1990.

Ohuchida et al., J. Am. Chem. Soc., 103(15):4597-4599, 198 1.

Oi and Morrison, Mt. Sinai J. Med., 53(3):175-180, 1986.

Osborn et al., Cell, 59:1203-1211, 1989.

Osterud et al., Thrombosis Res., 42:323-329, 1986.

Paborsky et al., J. Biol. Chem., 266(32):21911-21916, 1991.

Palleroni et al., Int. J. Cancer, 49:296-302, 1991.

Pasqualini et al., Nat. Biotechnol. 15:542-546, 1997.

Paulus, 1985

Perez et al., Nature, 316:354-356, 1985.

Perez et al., J. Immunol., 137:2069-2072, 1986a.

Perez et al., J. Exp. Med., 163:166-178, 1986b.

Pieterez et al., Antibody Immunoconj. Radiopharm., 1:79-103, 35, 1988.

Pimm et al., J. Cancer Res. Clin. Oncol., 118:367-370, 1992.

Pober et al., J. Exp. Med., 157:1339-1353, 1991.

Pukrittayakamee et al., Mol. Biol. Med., 1:123-135, 1983.

Qian et al., Cancer Res., 140:3250, 1991.

Rehemtulla et al., Thrombosis and Haemostatis. 65(5):521-527, 1991.

Reisfeld et al., Melanoma Antigens and Antibodies, p. 317, 1982.

Remington's Pharmaceutical Sciences, 16th Ed., Mack Publishing Company,1980.

Rettig et al., Proc. Natl. Acad. Sci. USA., 89:10832-10836, 1992.

Rivoltini et al., 3rd Int. Conf. Bispecific Antibodies and TargetedCellular Cytotoxicity, 1992.

Ruco et al., Am. J. Pathol., 137(5):1163-1171, 1990.

Ruf and Edgington, Proc. Natl. Acad. Sci. USA., 88:8430-8434, 1991a.

Ruf and Edgington, Thrombosis and Haemostasis, 66(5):529-533, 40, 1991b.

Ruf, et al., J. Biol. Chem., 266, pg. 2158, 1991.

Ruf and Edgington, FASEB J., 8:385-390, 1994.

Ruf et al., J. Biol. Chem., 267:22206-22210, 1992a.

Ruf et al., J. Biol. Chem., 267:6375-6381, 1992b.

Ruf et al., J. Biol. Chem., 267(31):22206-22210, 1992c.

Sakai and Kisiel, Thrombosis Res., 60:213-222, 1990.

Sambrook et al., Molecular Cloning, A Laboratory Manual., Cold SpringHarbor Laboratory, N.Y., 1989.

Sands, Immunoconjugates and Radiopharmaceuticals, 1:213-226, 1988.

Schutt et al., Immunol. Lett., 19:321-328, 1988.

Shen and Tai, J. Biol. Chem., 261(25):11585-11591, 1986.

Segal et al., Int. J. Cancer Suppl., 7 p36-8, 1992

Shankar et al., J. Biol. Chem., 269(19): 13936-13941, 1994.

Shepard et al., J. Clin. Immunol., 11 :117-127, 1991.

Shockley et al., Ann. N.Y. Acad. Sci., 617:367-382, 1991.

Smith et al., Br. J. Cancer, 59 (2) p 174-8, 1989.

Spiegelberg and Weigle, J. Exp. Med., 121:323-338, 1965.

Staerz et al., Nature, 314(6012):628-631, 1985.

Stevenson et al., Chem. Immunol, 48:126-166, 1990.

Stone, et al., Biochem J., 310:605, 1995.

Street et al., Cell. Immunol., 120:75-81, 1989.

Sugama et al., Jpn. J. Pharmacol., 55:2, pp. 287-290, 1991.

Sugama et al., J. Cell. Biol., 119(4):935-944, 1992.

ten Cate et al., J. Clin. Invest., 92:1207-1212, 1993.

Thieme et al., Diabetes, 44(1):98-103, 1995.

Thor et al., Cancer Res., 46:3118, 1986.

Ting et al., J. Immunol., 141:741-748, 1988.

Titus et al., J. Immunol., 138:4018-4022, 1987.

Tomiyama et al., Blood., 79(9):2303-2312, 1992.

Tone 1977

Tuttet al., Eur. J. Immunol., 21:1351-1358, 1991.

U.S. Pat. No. 4,196,265

U.S. Pat. No. 4,554,101

U.S. Pat. No. 4,975,369

U.S. Pat. No. 5,017,556

U.S. Pat. No. 5,110,730

U.S. Pat. No. 5,139,941

U.S. Pat. No. 5,183,756

U.S. Pat. No. 5,223,427

U.S. Pat. No. 5,242,813

U.S. Pat. No. 5,288,641

U.S. Pat. No. 5,346,991

U.S. Pat. No. 5,374,617

U.S. Pat. No. 5,437,864

U.S. Pat. No. 5,504,064

U.S. Pat. No. 5,504,067

U.S. Pat. No. 5,589,173

U.S. Pat. No. 5,589,363

Ugarova et al., J. Biol. Chem., 268(28):21080-21087, 1993.

Vaickus et al., Cancer Invest., 9:195-209, 1991.

Van Duk et al., Int. J. Cancer, 43:344-349, 1989.

Venkateswaran et al., Hybridoma, 11(6):729-739, 1992.

Vitetta et al., Cancer Res., 15:4052-4058, 1991.

Wang et al., Biochem. and Biophys. Res. Comm., 177(1):286-291, 1991.

Wang et al., Int. J. Cancer, 54:363-370, 1993.

Warret al., Blood, 75:1481-1489, 1990.

Watanbe et al., Proc. Natl. Acad. Sci. USA, 86:9456-9460, 1989.

Wawrzynczak and Thorpe In: Immunoconjugates: Antibody Conjugates inRadioimaging and Therapy of Cancer, Vogel (ed.), New York, OxfordUniversity Press, pp. 28-55, 1987.

Weiss et al., Blood, 73:968-975, 1989.

Whittle et al., Nature, 292:472-474, 1981.

Wildgoose et al., Blood, 80:25-28, 1992.

Williams and Esnouf, Biochem. J, 84:52-62, 1962.

Wiman and Collen, Eur. J. Biochem., 78:19-26, 1977.

Wiman, Biochem. J., 191:229-232, 1980.

Winter and Milstein, Nature, 349:293-299, 1991.

WO 94/05328, PCT Application

WO 94/07515, PCT Application

WO 94/28017, PCT Application

WO 96/01653, PCT Application

Wuet al., Int. J. Pharm., 12:235-239, 1990.

Xu et al., J. Biol. Chem., 267(25):17792-17803, 1992.

Yamaue et al., Biotherapy, 2:247-259, 1990.

Zamarron et al., J. Biol. Chem., 266(24):16193-16199, 1991.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 27                                            - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 220 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                 - Gly Ser Gly Thr Thr Asn Thr Val Ala Ala Ty - #r Asn Leu Thr Trp Lys         #                15                                                           - Ser Thr Asn Phe Lys Thr Ile Leu Glu Trp Gl - #u Pro Lys Pro Val Asn         #            30                                                               - Gln Val Tyr Thr Val Gln Ile Ser Thr Lys Se - #r Gly Asp Trp Lys Ser         #        45                                                                   - Lys Cys Phe Tyr Thr Thr Asp Thr Glu Cys As - #p Leu Thr Asp Glu Ile         #    60                                                                       - Val Lys Asp Val Lys Gln Thr Tyr Leu Ala Ar - #g Val Phe Ser Tyr Pro         #80                                                                           - Ala Gly Asn Val Glu Ser Thr Gly Ser Ala Gl - #y Glu Pro Leu Tyr Glu         #                95                                                           - Asn Ser Pro Glu Phe Thr Pro Tyr Leu Glu Th - #r Asn Leu Gly Gln Pro         #           110                                                               - Thr Ile Gln Ser Phe Glu Gln Val Gly Thr Ly - #s Val Asn Val Thr Val         #       125                                                                   - Glu Asp Glu Arg Thr Leu Val Arg Arg Asn As - #n Thr Phe Leu Ser Leu         #   140                                                                       - Arg Asp Val Phe Gly Lys Asp Leu Ile Tyr Th - #r Leu Tyr Tyr Trp Lys         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Ser Ser Ser Ser Gly Lys Lys Thr Ala Lys Th - #r Asn Thr Asn Glu Phe         #               175                                                           - Leu Ile Asp Val Asp Lys Gly Glu Asn Tyr Cy - #s Phe Ser Val Gln Ala         #           190                                                               - Val Ile Pro Ser Arg Thr Val Asn Arg Lys Se - #r Thr Asp Ser Pro Val         #       205                                                                   - Glu Cys Met Gly Gln Glu Lys Gly Glu Phe Ar - #g Glu                         #   220                                                                       - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 234 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                 - His His His His His His Ala Met Ala Leu Va - #l Pro Arg Gly Ser Cys         #                15                                                           - Gly Thr Thr Asn Thr Val Ala Ala Tyr Asn Le - #u Thr Trp Lys Ser Thr         #            30                                                               - Asn Phe Lys Thr Ile Leu Glu Trp Glu Pro Ly - #s Pro Val Asn Gln Val         #        45                                                                   - Tyr Thr Val Gln Ile Ser Thr Lys Ser Gly As - #p Trp Lys Ser Lys Cys         #    60                                                                       - Phe Tyr Thr Thr Asp Thr Glu Cys Asp Leu Th - #r Asp Glu Ile Val Lys         #80                                                                           - Asp Val Lys Gln Thr Tyr Leu Ala Arg Val Ph - #e Ser Tyr Pro Ala Gly         #                95                                                           - Asn Val Glu Ser Thr Gly Ser Ala Gly Glu Pr - #o Leu Tyr Glu Asn Ser         #           110                                                               - Pro Glu Phe Thr Pro Tyr Leu Glu Thr Asn Le - #u Gly Gln Pro Thr Ile         #       125                                                                   - Gln Ser Phe Glu Gln Val Gly Thr Lys Val As - #n Val Thr Val Glu Asp         #   140                                                                       - Glu Arg Thr Leu Val Arg Arg Asn Asn Thr Ph - #e Leu Ser Leu Arg Asp         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Val Phe Gly Lys Asp Leu Ile Tyr Thr Leu Ty - #r Tyr Trp Lys Ser Ser         #               175                                                           - Ser Ser Gly Lys Lys Thr Ala Lys Thr Asn Th - #r Asn Glu Phe Leu Ile         #           190                                                               - Asp Val Asp Lys Gly Glu Asn Tyr Cys Phe Se - #r Val Gln Ala Val Ile         #       205                                                                   - Pro Ser Arg Thr Val Asn Arg Lys Ser Thr As - #p Ser Pro Val Glu Cys         #   220                                                                       - Met Gly Gln Glu Lys Gly Glu Phe Arg Glu                                     225                 2 - #30                                                   - (2) INFORMATION FOR SEQ ID NO:3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 234 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                 - His His His His His His Ala Met Ala Leu Va - #l Pro Arg Gly Ser Gly         #                15                                                           - Thr Thr Asn Thr Val Ala Ala Tyr Asn Leu Th - #r Trp Lys Ser Thr Asn         #            30                                                               - Phe Lys Thr Ile Leu Glu Trp Glu Pro Lys Pr - #o Val Asn Gln Val Tyr         #        45                                                                   - Thr Val Gln Ile Ser Thr Lys Ser Gly Asp Tr - #p Lys Ser Lys Cys Phe         #    60                                                                       - Tyr Thr Thr Asp Thr Glu Cys Asp Leu Thr As - #p Glu Ile Val Lys Asp         #80                                                                           - Val Lys Gln Thr Tyr Leu Ala Arg Val Phe Se - #r Tyr Pro Ala Gly Asn         #                95                                                           - Val Glu Ser Thr Gly Ser Ala Gly Glu Pro Le - #u Tyr Glu Asn Ser Pro         #           110                                                               - Glu Phe Thr Pro Tyr Leu Glu Thr Asn Leu Gl - #y Gln Pro Thr Ile Gln         #       125                                                                   - Ser Phe Glu Gln Val Gly Thr Lys Val Asn Va - #l Thr Val Glu Asp Glu         #   140                                                                       - Arg Thr Leu Val Arg Arg Asn Asn Thr Phe Le - #u Ser Leu Arg Asp Val         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Phe Gly Lys Asp Leu Ile Tyr Thr Leu Tyr Ty - #r Trp Lys Ser Ser Ser         #               175                                                           - Ser Gly Lys Lys Thr Ala Lys Thr Asn Thr As - #n Glu Phe Leu Ile Asp         #           190                                                               - Val Asp Lys Gly Glu Asn Tyr Cys Phe Ser Va - #l Gln Ala Val Ile Pro         #       205                                                                   - Ser Arg Thr Val Asn Arg Lys Ser Thr Asp Se - #r Pro Val Glu Cys Met         #   220                                                                       - Gly Gln Glu Lys Gly Glu Phe Arg Glu Cys                                     225                 2 - #30                                                   - (2) INFORMATION FOR SEQ ID NO:4:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 220 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                 - Ser Cys Gly Thr Thr Asn Thr Val Ala Ala Ty - #r Asn Leu Thr Trp Lys         #                15                                                           - Ser Thr Asn Phe Lys Thr Ile Leu Glu Trp Gl - #u Pro Lys Pro Val Asn         #            30                                                               - Gln Val Tyr Thr Val Gln Ile Ser Thr Lys Se - #r Gly Asp Trp Lys Ser         #        45                                                                   - Lys Cys Phe Tyr Thr Thr Asp Thr Glu Cys As - #p Leu Thr Asp Glu Ile         #    60                                                                       - Val Lys Asp Val Lys Gln Thr Tyr Leu Ala Ar - #g Val Phe Ser Tyr Pro         #80                                                                           - Ala Gly Asn Val Glu Ser Thr Gly Ser Ala Gl - #y Glu Pro Leu Tyr Glu         #                95                                                           - Asn Ser Pro Glu Phe Thr Pro Tyr Leu Glu Th - #r Asn Leu Gly Gln Pro         #           110                                                               - Thr Ile Gln Ser Phe Glu Gln Val Gly Thr Ly - #s Val Asn Val Thr Val         #       125                                                                   - Glu Asp Glu Arg Thr Leu Val Arg Arg Asn As - #n Thr Phe Leu Ser Leu         #   140                                                                       - Arg Asp Val Phe Gly Lys Asp Leu Ile Tyr Th - #r Leu Tyr Tyr Trp Lys         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Ser Ser Ser Ser Gly Lys Lys Thr Ala Lys Th - #r Asn Thr Asn Glu Phe         #               175                                                           - Leu Ile Asp Val Asp Lys Gly Glu Asn Tyr Cy - #s Phe Ser Val Gln Ala         #           190                                                               - Val Ile Pro Ser Arg Thr Val Asn Arg Lys Se - #r Thr Asp Ser Pro Val         #       205                                                                   - Glu Cys Met Gly Gln Glu Lys Gly Glu Phe Ar - #g Glu                         #   220                                                                       - (2) INFORMATION FOR SEQ ID NO:5:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 220 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                 - Ser Gly Thr Thr Asn Thr Val Ala Ala Tyr As - #n Leu Thr Trp Lys Ser         #                15                                                           - Thr Asn Phe Lys Thr Ile Leu Glu Trp Glu Pr - #o Lys Pro Val Asn Gln         #            30                                                               - Val Tyr Thr Val Gln Ile Ser Thr Lys Ser Gl - #y Asp Trp Lys Ser Lys         #        45                                                                   - Cys Phe Tyr Thr Thr Asp Thr Glu Cys Asp Le - #u Thr Asp Glu Ile Val         #    60                                                                       - Lys Asp Val Lys Gln Thr Tyr Leu Ala Arg Va - #l Phe Ser Tyr Pro Ala         #80                                                                           - Gly Asn Val Glu Ser Thr Gly Ser Ala Gly Gl - #u Pro Leu Tyr Glu Asn         #                95                                                           - Ser Pro Glu Phe Thr Pro Tyr Leu Glu Thr As - #n Leu Gly Gln Pro Thr         #           110                                                               - Ile Gln Ser Phe Glu Gln Val Gly Thr Lys Va - #l Asn Val Thr Val Glu         #       125                                                                   - Asp Glu Arg Thr Leu Val Arg Arg Asn Asn Th - #r Phe Leu Ser Leu Arg         #   140                                                                       - Asp Val Phe Gly Lys Asp Leu Ile Tyr Thr Le - #u Tyr Tyr Trp Lys Ser         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Ser Ser Ser Gly Lys Lys Thr Ala Lys Thr As - #n Thr Asn Glu Phe Leu         #               175                                                           - Ile Asp Val Asp Lys Gly Glu Asn Tyr Cys Ph - #e Ser Val Gln Ala Val         #           190                                                               - Ile Pro Ser Arg Thr Val Asn Arg Lys Ser Th - #r Asp Ser Pro Val Glu         #       205                                                                   - Cys Met Gly Gln Glu Lys Gly Glu Phe Arg Gl - #u Cys                         #   220                                                                       - (2) INFORMATION FOR SEQ ID NO:6:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 235 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                 - His His His His His His Ala Met Ala Leu Va - #l Pro Arg Gly Ser Gly         #                15                                                           - Thr Thr Asn Thr Val Ala Ala Tyr Asn Leu Th - #r Trp Lys Ser Thr Asn         #            30                                                               - Phe Lys Thr Ile Leu Glu Trp Glu Pro Lys Pr - #o Val Asn Gln Val Tyr         #        45                                                                   - Thr Val Gln Ile Ser Thr Lys Ser Gly Asp Tr - #p Lys Ser Lys Cys Phe         #    60                                                                       - Tyr Thr Thr Asp Thr Glu Cys Asp Leu Thr As - #p Glu Ile Val Lys Asp         #80                                                                           - Val Lys Gln Thr Tyr Leu Ala Arg Val Phe Se - #r Tyr Pro Ala Gly Asn         #                95                                                           - Val Glu Ser Thr Gly Ser Ala Gly Glu Pro Le - #u Tyr Glu Asn Ser Pro         #           110                                                               - Glu Phe Thr Pro Tyr Leu Glu Thr Asn Leu Gl - #y Gln Pro Thr Ile Gln         #       125                                                                   - Ser Phe Glu Gln Val Gly Thr Lys Val Asn Va - #l Thr Val Glu Asp Glu         #   140                                                                       - Arg Thr Leu Val Arg Arg Asn Asn Thr Phe Le - #u Ser Leu Arg Asp Val         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Phe Gly Lys Asp Leu Ile Tyr Thr Leu Tyr Ty - #r Trp Lys Ser Ser Ser         #               175                                                           - Ser Gly Lys Lys Thr Ala Lys Thr Asn Thr As - #n Glu Phe Leu Ile Asp         #           190                                                               - Val Asp Lys Gly Glu Asn Tyr Cys Phe Ser Va - #l Gln Ala Val Ile Pro         #       205                                                                   - Ser Arg Thr Val Asn Arg Lys Ser Thr Asp Se - #r Pro Val Glu Cys Met         #   220                                                                       - Gly Gln Glu Lys Gly Glu Phe Arg Glu Ile Cy - #s                             225                 2 - #30                 2 - #35                           - (2) INFORMATION FOR SEQ ID NO:7:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 236 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                 - His His His His His His Ala Met Ala Leu Va - #l Pro Arg Gly Ser Gly         #                15                                                           - Thr Thr Asn Thr Val Ala Ala Tyr Asn Leu Th - #r Trp Lys Ser Thr Asn         #            30                                                               - Phe Lys Thr Ile Leu Glu Trp Glu Pro Lys Pr - #o Val Asn Gln Val Tyr         #        45                                                                   - Thr Val Gln Ile Ser Thr Lys Ser Gly Asp Tr - #p Lys Ser Lys Cys Phe         #    60                                                                       - Tyr Thr Thr Asp Thr Glu Cys Asp Leu Thr As - #p Glu Ile Val Lys Asp         #80                                                                           - Val Lys Gln Thr Tyr Leu Ala Arg Val Phe Se - #r Tyr Pro Ala Gly Asn         #                95                                                           - Val Glu Ser Thr Gly Ser Ala Gly Glu Pro Le - #u Tyr Glu Asn Ser Pro         #           110                                                               - Glu Phe Thr Pro Tyr Leu Glu Thr Asn Leu Gl - #y Gln Pro Thr Ile Gln         #       125                                                                   - Ser Phe Glu Gln Val Gly Thr Lys Val Asn Va - #l Thr Val Glu Asp Glu         #   140                                                                       - Arg Thr Leu Val Arg Arg Asn Asn Thr Phe Le - #u Ser Leu Arg Asp Val         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Phe Gly Lys Asp Leu Ile Tyr Thr Leu Tyr Ty - #r Trp Lys Ser Ser Ser         #               175                                                           - Ser Gly Lys Lys Thr Ala Lys Thr Asn Thr As - #n Glu Phe Leu Ile Asp         #           190                                                               - Val Asp Lys Gly Glu Asn Tyr Cys Phe Ser Va - #l Gln Ala Val Ile Pro         #       205                                                                   - Ser Arg Thr Val Asn Arg Lys Ser Thr Asp Se - #r Pro Val Glu Cys Met         #   220                                                                       - Gly Gln Glu Lys Gly Glu Phe Arg Glu Ile Ph - #e Cys                         225                 2 - #30                 2 - #35                           - (2) INFORMATION FOR SEQ ID NO:8:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 219 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                 - Ser Gly Thr Thr Asn Thr Val Ala Ala Tyr As - #n Leu Thr Trp Lys Ser         #                15                                                           - Thr Asn Phe Lys Thr Ile Leu Glu Trp Glu Pr - #o Lys Pro Val Asn Gln         #            30                                                               - Val Tyr Thr Val Gln Ile Ser Thr Lys Ser Gl - #y Asp Trp Lys Ser Lys         #        45                                                                   - Cys Phe Tyr Thr Thr Asp Thr Glu Cys Asp Le - #u Thr Asp Glu Ile Val         #    60                                                                       - Lys Asp Val Lys Gln Thr Tyr Leu Ala Arg Va - #l Phe Ser Tyr Pro Ala         #80                                                                           - Gly Asn Val Glu Ser Thr Gly Ser Ala Gly Gl - #u Pro Leu Tyr Glu Asn         #                95                                                           - Ser Pro Glu Phe Thr Pro Tyr Leu Glu Thr As - #n Leu Gly Gln Pro Thr         #           110                                                               - Ile Gln Ser Phe Glu Gln Val Gly Thr Lys Va - #l Asn Val Thr Val Glu         #       125                                                                   - Asp Glu Arg Thr Leu Val Arg Arg Asn Asn Th - #r Phe Leu Ser Leu Arg         #   140                                                                       - Asp Val Phe Gly Lys Asp Leu Ile Tyr Thr Le - #u Tyr Tyr Arg Lys Ser         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Ser Ser Ser Gly Lys Lys Thr Ala Lys Thr As - #n Thr Asn Glu Phe Leu         #               175                                                           - Ile Asp Val Asp Lys Gly Glu Asn Tyr Cys Ph - #e Ser Val Gln Ala Val         #           190                                                               - Ile Pro Ser Arg Thr Val Asn Arg Lys Ser Th - #r Asp Ser Pro Val Glu         #       205                                                                   - Cys Met Gly Gln Glu Lys Gly Glu Phe Arg Gl - #u                             #   215                                                                       - (2) INFORMATION FOR SEQ ID NO:9:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 219 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                 - Ser Gly Thr Thr Asn Thr Val Ala Ala Tyr As - #n Leu Thr Trp Lys Ser         #                15                                                           - Thr Asn Phe Lys Thr Ile Leu Glu Trp Glu Pr - #o Lys Pro Val Asn Gln         #            30                                                               - Val Tyr Thr Val Gln Ile Ser Thr Lys Ser Gl - #y Asp Trp Lys Ser Lys         #        45                                                                   - Cys Phe Tyr Thr Thr Asp Thr Glu Cys Asp Le - #u Thr Asp Glu Ile Val         #    60                                                                       - Lys Asp Val Lys Gln Thr Tyr Leu Ala Arg Va - #l Phe Ser Tyr Pro Ala         #80                                                                           - Gly Asn Val Glu Ser Thr Gly Ser Ala Gly Gl - #u Pro Leu Tyr Glu Asn         #                95                                                           - Ser Pro Glu Phe Thr Pro Tyr Leu Glu Thr As - #n Leu Gly Gln Pro Thr         #           110                                                               - Ile Gln Ser Phe Glu Gln Val Gly Thr Lys Va - #l Asn Val Thr Val Glu         #       125                                                                   - Asp Glu Arg Thr Leu Val Arg Arg Asn Asn Th - #r Phe Leu Ser Leu Arg         #   140                                                                       - Asp Val Phe Gly Lys Asp Leu Ile Tyr Thr Le - #u Tyr Tyr Trp Lys Ser         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Ser Ser Ser Ala Lys Lys Thr Ala Lys Thr As - #n Thr Asn Glu Phe Leu         #               175                                                           - Ile Asp Val Asp Lys Gly Glu Asn Tyr Cys Ph - #e Ser Val Gln Ala Val         #           190                                                               - Ile Pro Ser Arg Thr Val Asn Arg Lys Ser Th - #r Asp Ser Pro Val Glu         #       205                                                                   - Cys Met Gly Gln Glu Lys Gly Glu Phe Arg Gl - #u                             #   215                                                                       - (2) INFORMATION FOR SEQ ID NO:10:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 657 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                - TCAGGCACTA CAAATACTGT GGCAGCATAT AATTTAACTT GGAAATCAAC TA - #ATTTCAAG         60                                                                          - ACAATTTTGG AGTGGGAACC CAAACCCGTC AATCAAGTCT ACACTGTTCA AA - #TAAGCACT        120                                                                          - AAGTCAGGAG ATTGGAAAAG CAAATGCTTT TACACAACAG ACACAGAGTG TG - #ACCTCACC        180                                                                          - GACGAGATTG TGAAGGATGT GAAGCAGACG TACTTGGCAC GGGTCTTCTC CT - #ACCCGGCA        240                                                                          - GGGAATGTGG AGAGCACCGG TTCTGCTGGG GAGCCTCTGT ATGAGAACTC CC - #CAGAGTTC        300                                                                          - ACACCTTACC TGGAGACAAA CCTCGGACAG CCAACAATTC AGAGTTTTGA AC - #AGGTGGGA        360                                                                          - ACAAAAGTGA ATGTGACCGT AGAAGATGAA CGGACTTTAG TCAGAAGGAA CA - #ACACTTTC        420                                                                          - CTAAGCCTCC GGGATGTTTT TGGCAAGGAC TTAATTTATA CACTTTATTA TT - #GGAAATCT        480                                                                          - TCAAGTTCAG GAAAGAAAAC AGCCAAAACA AACACTAATG AGTTTTTGAT TG - #ATGTGGAT        540                                                                          - AAAGGAGAAA ACTACTGTTT CAGTGTTCAA GCAGTGATTC CCTCCCGAAC AG - #TTAACCGG        600                                                                          - AAGAGTACAG ACAGCCCGGT AGAGTGTATG GGCCAGGAGA AAGGGGAATT CA - #GAGAA           657                                                                          - (2) INFORMATION FOR SEQ ID NO:11:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 13865 base                                                        (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                - GAATTCTCCC AGAGGCAAAC TGCCAGATGT GAGGCTGCTC TTCCTCAGTC AC - #TATCTCTG         60                                                                          - GTCGTACCGG GCGATGCCTG AGCCAACTGA CCCTCAGACC TGTGAGCCGA GC - #CGGTCACA        120                                                                          - CCGTGGCTGA CACCGGCATT CCCACCGCCT TTCTCCTGTG CGACCCGCTA AG - #GGCCCCGC        180                                                                          - GAGGTGGGCA GGCCAAGTAT TCTTGACCTT CGTGGGGTAG AAGAAGCCAC CG - #TGGCTGGG        240                                                                          - AGAGGGCCCT GCTCACAGCC ACACGTTTAC TTCGCTGCAG GTCCCGAGCT TC - #TGCCCCAG        300                                                                          - GTGGGCAAAG CATCCGGGAA ATGCCCTCCG CTGCCCGAGG GGAGCCCAGA GC - #CCGTGCTT        360                                                                          - TCTATTAAAT GTTGTAAATG CCGCCTCTCC CACTTTATCA CCAAATGGAA GG - #GAAGAATT        420                                                                          - CTTCCAAGGC GCCCTCCCTT TCCTGCCATA GACCTGCAAC CCACCTAAGC TG - #CACGTCGG        480                                                                          - AGTCGCGGGC CTGGGTGAAT CCGGGGGCCT TGGGGGACCC GGGCAACTAG AC - #CCGCCTGC        540                                                                          - GTCCTCCAGG GCAGCTCCGC GCTCGGTGGC GCGGTTGAAT CACTGGGGTG AG - #TCATCCCT        600                                                                          - TGCAGGGTCC CGGAGTTTCC TACCGGGAGG AGGCGGGGCA GGGGTGTGGA CT - #CGCCGGGG        660                                                                          - GCCGCCCACC GCGACGGCAA GTGACCCGGG CCGGGGGCGG GGAGTCGGGA GG - #AGCGGCGG        720                                                                          - GGGCGGGCGC CGGGGGCGGG CAGAGGCGCG GGAGAGCGCG CCGCCGGCCC TT - #TATAGCGC        780                                                                          - GCGGGGCACC GGCTCCCCAA GACTGCGAGC TCCCCGCACC CCCTCGCACT CC - #CTCTGGCC        840                                                                          - GGCCCAGGGC GCCTTCAGCC CAACCTCCCC AGCCCCACGG GCGCCACGGA AC - #CCGCTCGA        900                                                                          - TCTCGCCGCC AACTGGTAGA CATGGAGACC CCTGCCTGGC CCCGGGTCCC GC - #GCCCCGAG        960                                                                          - ACCGCCGTCG CTCGGACGCT CCTGCTCGGC TGGGTCTTCG CCCAGGTGGC CG - #GCGCTTCA       1020                                                                          - GGTGAGTGGC ACCAGCCCCT GGAAGCCCGG GGCGCGCCAC ACGCAGGAGG GA - #GGCGACAG       1080                                                                          - TCCTGGCTGG CAGCGGGCTC GCCCTGGTTC CCCGGGGCGC CCATGTTGTC CC - #CCGCGCCT       1140                                                                          - ACGGGACTCG GCTGCGCTCA CCCAGCCCGG CTTGAATGAA CCGAGTCCGT CG - #GGCGCCGG       1200                                                                          - CGGGAGTTGC AGGGAGGGAG TTGGCGCCCC AGACCCCGCT GCCCCTTCCG CT - #GGAGAGTT       1260                                                                          - TTGCTCGGGG TGTCCGAGTA ATTGGACTGT TGTTGCATAA GCGGACTTTT AG - #CTCCCGCT       1320                                                                          - TTAACTCTGG GGAAAGGGCT TCCCAGTGAG TTGCGACCTT CAATATGATA GG - #ACTTGTGC       1380                                                                          - CTGCGTCTGC ACGTGTTGGC GTGCAGAGGT TTGGATATTA TCTTTCATTA TA - #TGTGCATC       1440                                                                          - TTCCCTTAAT AAAGAGCGTC CCTGGTCTTT TCCTGGCCAT CTTTGTTCTA GG - #TTTGGGTA       1500                                                                          - GAGGCAATCC AAAAGGGCTG GATTGCTGCT TAGATTGGAG CAGGTACAAC GT - #TGTGCATG       1560                                                                          - CCCCGTATTT CTACGAGGTG TTCGGGACGG CGTAGAGACT GGGACCTGCT GC - #GTACTGGC       1620                                                                          - AAAGCAGACC TTCATAAGAA ATAATCCTGA TCCAATACAG CCGACGGTGT GA - #CAGGCCAC       1680                                                                          - ACGTCCCCGT GGGTCTCTGT GGAAGTTTCA GTGTAGCGAC ATTTCAGATA AA - #AGTGGAAA       1740                                                                          - AAGTGAAGTT TGGCTTTTTT CATTTGTATG CAGTCCTAAC TCTTGTCACA CG - #TGTGGGAT       1800                                                                          - TTATCTTTTT CCATAACTTA CTGAAAACCC TTCCTGGCGG GCTGAACCTG AC - #TCTTCCTG       1860                                                                          - AGCTGAGTCC TGGACTGGCA CACTGATGGC TCTGGGCTCT TCCCGGTCAA GT - #TATAACAA       1920                                                                          - GGCTTTGCCC ATGAATAATT TCAAACGAAA ATGTCAAGAT CCTTGCCGGT GT - #CCTGGGAT       1980                                                                          - TACAAGGTGA ATCTTGTCAT GAAGAAATTC TAGGTCTAGA AAAAATTTGA AG - #ATTCTTTT       2040                                                                          - TCTCTTGATA ATTCACTAAT GAAGCTTTTG TGGTTGAAAA ATAAAAAGTG AG - #GTTTATGG       2100                                                                          - TGATGTCAGG TGGGAAGGTG TTTTATACAT CAATACATTC GAGTGCTCTG AA - #GTGCATGT       2160                                                                          - AATAATAGCT GTTTCTCTGT TGTTTAAAGG CACTACAAAT ACTGTGGCAG CA - #TATAATTT       2220                                                                          - AACTTGGAAA TCAACTAATT TCAAGACAAT TTTGGAGTGG GAACCCAAAC CC - #GTCAATCA       2280                                                                          - AGTCTACACT GTTCAAATAA GGTAAGCTGG GTACAGAAAA AGAAAATTAA GG - #TCTTTGAT       2340                                                                          - GTTTCTACTG TCCTATGCTG AACAAGAATG TCTTTAAAGC TGATTACTGG AT - #GAAATTAT       2400                                                                          - TTAACAGATG ACGAAGAAGA AGGGATTCTT GGCAATTCGC TGGCCGGTGT CA - #TACTCTAT       2460                                                                          - TAGGCCTGCA ACATTTCCAG ACCTTAAACT GATAGAACAT TTTAATTGTT TT - #AATTGTTT       2520                                                                          - TTGGAAATGA TGGGAGAGTT CCTAAGTGGA GTATAAACTG TGGAGAGATG AA - #CCATCTTG       2580                                                                          - AGTAGGCACT GAAGTGTGCT TTGGGTCATG ATAGATTAAT TAATCTCATC TA - #AACATTGA       2640                                                                          - TGTCTTTTTC CGTTGCTGTC TAGACTGTGA ACAATGTCTA ACACCTTAGG GA - #AGAGGTGG       2700                                                                          - GGAGGAATCC CAATGTATAC ATTGCCCTTA AGCAGTGTTT GATTCATTCA TC - #TTTGGACT       2760                                                                          - CCATGAATCG AAATCTGGTA GAATACATGA TCTTAGTGGA GGAGGCCAAA TG - #CGTGACTC       2820                                                                          - ACTGAGCCTG GCAGAGCAGA AATACTCTGC TGTCTGCACC CTCTGGGTCT GG - #TGTGGCTC       2880                                                                          - TGCTTCTTGG TGCTTCAACT CTGACTGGCA GCTGTCCCCA GGAGGCGATA AT - #TCAGCATG       2940                                                                          - TTCAATCTAA AGGTTATGAC TTCCTTGATG GTTTTCACCA TATTCTTGGC AA - #GTTTTTGG       3000                                                                          - TTTTTGAAAT GTTCTAGGAG GCTTGGTAGA GATCTTATGA AATAGAGAAT AG - #CTGCTGTG       3060                                                                          - GAAATTATTT TAATGCTAAT TACATAAAAG TACAAAAGTA GCACTAGCTA AA - #ACAAAAGG       3120                                                                          - TATTTTGCTG TTCTGTTTTG TTTTAGCTTG TGCCAGGCCT TTTACAGCAT TA - #GGAATGCA       3180                                                                          - ACTTCTAGAT AACGATGCAT CTTTTAAGTG AATGTTCTTG TTTTTCAAAA TG - #AACTTCAT       3240                                                                          - GACAGTAGTT GCCAAACCAG CAAGGAGAAC TTGCATGCAT ACGTGCATGC AT - #GTGTGGAT       3300                                                                          - ATGTATGGGG GTGGGGGGAG AGAAAGATGA AGGAATTTCA TAACATGAAA TA - #ATGATTAC       3360                                                                          - AGTTCTGGTC AAACTTGTCA ATTCAGATTT CACCAATTGA GAATTAGTAA GT - #AATTTCTC       3420                                                                          - TGATACAGGC CTGAAGTTTA CCTTAGTAAA CACTTTACTT CCATATGGTA AA - #AATTAGAT       3480                                                                          - TTTGGGAGGA ATGCTTACCT CCTAAATATA TTCAATCTAA TATTTGAGGA CA - #CATGGGAA       3540                                                                          - TATATTTATG ATTCATCTGC TTTTTAAACA TAAGCCTTTG TTAACTGTAA GT - #TCTTGAAC       3600                                                                          - TTTATAAGGC TGCTGTTATT TAAATGAGCA CAGCTCCTGA TCTGCAAACA GC - #AGAGCGCA       3660                                                                          - GGGCTACAGC TTGGGGGATG CCAGCCGACT CAGGGTGGTC CTGTGGACTG AA - #CAATCTCT       3720                                                                          - TGCTGCTGTA CTGGAGGGCC TGGGAGCTTT TCCATCAGCC TCGGCCTGAG GT - #GTGCACTC       3780                                                                          - TTCTCCTGCC CACCCCAGGA ATAAATGAGA TTCCTGGTTA AAAAGGACCA GA - #GCAGTCAT       3840                                                                          - TTTACAGTTG AGGAAACTGT TGCTCTGAGA AGTGAGGGAT TTATTCATGA CT - #ACACTGAT       3900                                                                          - GGTGAGTGCC CATGTCAGGT CTGGAACCAA AGTCTACCCA GTATCCACAC AC - #CACCATCC       3960                                                                          - CTCAGGTGGC TCTGCCACAG TCTGATGGGA GGCTCCAAAG CGGGAGGAAG AA - #GGAAAGTC       4020                                                                          - TTGCCCACTG CATCTCCTCA GTTGGCCTTC CTCTCTGCCT GTTTTCCCTC CC - #TACAGTTA       4080                                                                          - GCATCTTAAG CAGCTGCCTC TCTTCCCTCC CGACTGCTCT CACTACTGCA GC - #CTGGCTCC       4140                                                                          - AGCCGCAGGA CACTACTGCT GTGCAGAAGC CCCTACTTGG AACTCCAACT GC - #ATTTTTCA       4200                                                                          - CCTTTGCTAA CAGTTTTCAG TGGTGGTTGG GAAATGTTAT TGGCTTAAGC CT - #TAGCACAA       4260                                                                          - ACCGTCACCG GTGATATTCA TTCCATGGAA ATGTTCTGAA TTCTAAAGCT GA - #ATTTACAA       4320                                                                          - AGCTTCTGGA AAACAACCTG CAACCAAATT AGTGACTGAA TTTTTTAGTT AA - #CTCAAAAT       4380                                                                          - TCCAAATCAG AGGGTTTTGC AATGCCTGGA GGAACCTTGG AGGCTTTTAA AG - #TGTTAATG       4440                                                                          - CTATTAATGG CATTCAGAGG GATTTTCTAC AGAATTGTCC CTTCATTACC TG - #TTTATACA       4500                                                                          - GTTTTACTAC TTACCAGGGT ACTGTATAAA TCCTTGTGCT AAATTTTGCT AT - #AGAGTATG       4560                                                                          - TGGTCCCTGC TGTGAGCTGG GAGGAACCAA ATACTGTATC TCTATGTTAC AT - #AGAAAGCC       4620                                                                          - CTAGGAGACT TTCTCCTGTT ATCTGAACAA CTATTTGCTG TACTGATAAA AA - #GGAAACAG       4680                                                                          - CATAGTCTCA TTCACTTTTT GAAATGGAAA TGATAAAATA AAACACATTT TG - #GTCATTCG       4740                                                                          - GGAACAAAAT ACCCTCTCTA CTTTTATCAC ATAAAATTAA ATAAATAGAA AC - #CAAAATAT       4800                                                                          - TTCAGTATCA ATCTTAGTTT GTGCACTTTA GGATAAAGAA TGTGTTTACC CA - #AATCCTTT       4860                                                                          - TGGCCTGGTT ACTTAGTTCA GATTTTGAAA GAAAATATAT TTGTGGCTTT TA - #TGTGTGAA       4920                                                                          - TTTAGACAAT GGAATCCATG TGGTGCCTCG TTTTCCCTGA GATTATGTAT TA - #ATTCAACC       4980                                                                          - TGTAAATGCA AACCATCTAA TAGTCAGCGA GACCCTATAG CCCTGCTGCT TA - #ATGGGGGC       5040                                                                          - ACACAAGGGC ATGCAGCCCT CGTACCAGGC AGACTGTGTT CATATTAACA GC - #ATCGTGGA       5100                                                                          - GAAACTCATG CTGGGGGACA GGGGAGGGAG ATGTAAATGC TCAGCAGGGA GA - #TCTGGAGA       5160                                                                          - TTCCTGGAGC AGGTGGAGTT GGGACCTGGC CTTGAACGAT GGGTCTGGCT CT - #GGCAGTCA       5220                                                                          - GTAATGCCAA AGGGAAGAGC AGCATAACTG TCACTTTCCA TGGGACAGAA GT - #GTGTGAAT       5280                                                                          - CAAGTTGCAG TGACGCTTCA CCTATTTATT ATTTTGGTCA TTTAGAAGAA TT - #TCATTGTC       5340                                                                          - AGTAGAAGTC CTTTAAATCA TTTCCCCTTC AGTGACGTCT CACAAAAAAA AG - #ATCTGTCT       5400                                                                          - TTAGCTTTTT AGTCTCAGAC TTTATTAGAC AGATACTACC TGTACTCTTA TT - #CTGTAATC       5460                                                                          - TTTGTTGGGA TGGATTCACA TCTTGCAAAG GAAGGGAGGC ATGTAGTATA AT - #GGGGCAAA       5520                                                                          - CAGACCCAGC TCTGCCACTC GTTAGATATG TGACCTTCTG CAAGTTGCTT AG - #TGCCTGTG       5580                                                                          - AGCTTCAGTG TCCTCATGGA TAAGAAAGAT CCAACACCTT CTTGGAAGGA TT - #ATATCAAA       5640                                                                          - TGAAGTAACA TGAGTAAAGG GTCCAGCAGA ATACCTGGCA TATAGTGGAG TC - #AATGAATG       5700                                                                          - ATTAATAATA TTATTAATAG TGGTCATGAG AGATATATGT ATAACATGTT AT - #TATGTAGA       5760                                                                          - CTCACTATAT AGACTCTATT CTACATAGAA TATAGAACAT TATATAACAA AC - #AACTATAA       5820                                                                          - TAAGTAGACT ATAGTAAACA ACCTCACTTT GTCTCAGTTG CCTCATCTTG AT - #GGAAAACT       5880                                                                          - GCTCTTTCTC TCCTGTTACC CTGACAGAGA GCGTCTACAT TCTAAAAGAA AG - #ATATTTAA       5940                                                                          - CAAAATGGTT GAGTACAGAT CCAAGAGTCA AATAGCTGTC TGGTTCAAAG TC - #CAGCTGTG       6000                                                                          - TGATTTTGAG CTAGTCACCC AATCTCACTT TGTCTCAGTA GCCTTATTTG TA - #AAAACAAG       6060                                                                          - GCAAATTACA GAGCCATCCC CTGGGTTGCT ATGAGGACTC AAACATGCAT CC - #CAAGTGCT       6120                                                                          - CGGTGTTGCT AGGTATGATG GCTCACACCT GTACATTCAG CACTTTGGGA GG - #CCGAAGCA       6180                                                                          - GAAGGATCAG CCTGGGCAAC ATAGCAGGAC CCCATCTCTA CAAAACAATG TT - #TAAAAAAA       6240                                                                          - AGCAAAGTGC TCAGCACAGT GACTGCATCA TTAGGATTGA TTGTAGGGCT CC - #TGATGTTA       6300                                                                          - GCACAGAACA CCACAGCCAG GAAGCAGTCT ATCTTGTTGG GTGCAAATTG TA - #ACATTCCA       6360                                                                          - TTTATGTTTC TTCCTTCTTT TCTTTCTTTA GCACTAAGTC AGGAGATTGG AA - #AAGCAAAT       6420                                                                          - GCTTTTACAC AACAGACACA GAGTGTGACC TCACCGACGA GATTGTGAAG GA - #TGTGAAGC       6480                                                                          - AGACGTACTT GGCACGGGTC TTCTCCTACC CGGCAGGGAA TGTGGAGAGC AC - #CGGTTCTG       6540                                                                          - CTGGGGAGCC TCTGTATGAG AACTCCCCAG AGTTCACACC TTACCTGGAG AG - #TAAGTGGC       6600                                                                          - TTGGGCTGTA ATACCGTTCA TTCTTGTTAG AAACGTCTGA ACATTCTCGT GA - #TCTTGTGC       6660                                                                          - CTTTAGGGGC TACAAAATTA AAAATATTTA TTCTTTTTTT CTCAGAAACT GG - #TATGTATC       6720                                                                          - ACAGCCCTCT TCACACATTC CAGATGTGGT AGGAGGTTCA CAGAATGTGA AC - #TTTTGGAG       6780                                                                          - CTGATGACAG TGTCATCAAG TAACTTTCTC CCCCAGTCTG TCCCCAGACC CT - #GTTACTGT       6840                                                                          - CCTCAGTAAG CGGCTGAATG TGTGTTGGGA GAGGGCGGGC CAGGGAAGCG GG - #TAGGGATA       6900                                                                          - GGAAATCCAC CAAGGCCGGG GTTTTAGCTT TTCCCTATAT ATATATCATG TA - #TCCTGATT       6960                                                                          - TTTCTGTCCC GTTATCACAC TAAAAATCCC AGTTGAGGAT TTTTCCCAAA CG - #GTCATAAA       7020                                                                          - TCAATGAGGA AAGTCCATGG TTTCCCTCTG AGCCCATAAT TAGCCTAATT AT - #GCTGACCT       7080                                                                          - TTTCTAATCA GTTGGCCATG ATTTGAGTTC CGTGATGTGC CAGCACCTGC CC - #AGCCATCT       7140                                                                          - GCCTGTCACC CTCGTTCTGG TTTTGGAAAG GTGGAATACT TTCCTCCTCA GC - #CTTTGCCC       7200                                                                          - CTGTAAGCTG GCCCTAGGAG CCAGTAAAAG AATGAAGAGA ATTCCTGTCA AG - #TAGGAGAT       7260                                                                          - TTATTCTTTT GCCGCAACTG TGGCTCTGAG CTAGGCAATT TAGATAAATG CA - #TGTAGCAC       7320                                                                          - ATTGAGTAGA GTGAAATTAG CTTCTCTTGT AAGGCCAGCT GGTTAGAATG AA - #GGTGTTGT       7380                                                                          - GTGAGTGTTA GGCCCAGCGA GAGAGAACAG TTTCTCAAGG TAGGAATGGT GA - #AAAGAAGG       7440                                                                          - GGTGGACGGA CAACCAACCA ACCATCCTCC TCTGGTATCT ACTTTGAGGG TT - #GAAATAGG       7500                                                                          - GGGCCTGACC CCAGGTGAAT GTGGCTGCCT TCCCAGAGCC CCCATTTGCA AG - #ACCCTCCA       7560                                                                          - GACCCCCAGG TGCTTCTGCT TGTGTCTTTT GTGGCACCAG GCAAGAATGT AG - #CAGCGTCA       7620                                                                          - GCAGCCCCTC TGGTGACTGT GGCATGGTTG ACATTCATTT CCCCCCTAAT TA - #ATGGCATC       7680                                                                          - CTCATGATTC TCTTTTATAT TAATAGTTCT TGAGTTTTTT TGTAAGCTAC TT - #CAAATCCT       7740                                                                          - TTGTTGGTGC AAGATAGAAG ATATTTTATG TGTTTGTTTT GCATGTGCAC AC - #ACATATTT       7800                                                                          - GGCCTGTGAA TTGATGTTTG TTTTCCTGTC ATTTAACCAA AGCACATGAG AT - #AATTGAGC       7860                                                                          - CATTGCAGAG ACCCCGTGGT TAAATCCGGC TTCTCGAGGT ACCAAGGACA TT - #TCCTGGGC       7920                                                                          - TTTCTCACAG CCCTACATAT TTTTGAACCT AAAATATCGT AGTTTATGCT AC - #CACCCTGT       7980                                                                          - TCAGTATAGT AGCCACTAGC CACATGTGGC TGTTGACCAC TTGAAATATG GC - #TAATGCTC       8040                                                                          - TAAGTATAAA GTACACACTG GAATTTAAGA AGTGTAGAAT ATCTCAAAAC TT - #TTTTATAT       8100                                                                          - TGATTACACA TTAAAATGAT TATATTCCAG ATATATGCAG TTGACTCAAG CA - #ATGCATGG       8160                                                                          - CTGAGAGGCA CCGACTCCCT GTGCAGTTGA AAATCCGAGT ATAACTTGAC TC - #CCCAAAAA       8220                                                                          - CTTAACTACT AATAGCCTAC CTATCGGTTG ACTGTTGACT GCAGCCTTAC CA - #ATAAGATA       8280                                                                          - AACAGTCAAT TAACACACAT TTTTCATGTT GCGTGTATTA TATACTGTAT TC - #TTACAATA       8340                                                                          - AAGTAAGCTA GAGGAAAGAA AATGTTATTA AGAAAATTAT AAGGAAAAGA GG - #CTGGGCAT       8400                                                                          - GGTGGCTCGT GCCTGTAATC TCAGAACTTT GGGATGCTAA GGCGGGTGGA TC - #ACTTGAGG       8460                                                                          - TCAGGAGTTC AAGACCAGCC TGGCCAACAT GGTGAAACCC CATCTCTACT AA - #AAATACAA       8520                                                                          - AAATTAGCCA GGCGTGGTTG TGGGTGCCTG TAATCCCAGC TACTTGGGAG GC - #TGAGGCAG       8580                                                                          - GAGAATCACT TCGACCCAGG TGGAGGAGGT TGCAGTGAAC TGAGATTGCG CC - #ACTGCACT       8640                                                                          - CCGGCCTGGG TGACAGAGCG AGACTCTGTC TAAAAAAGAA AGGGAAAGAA AG - #AAAAAAAA       8700                                                                          - GAAAAGAAAA GAAAAGAAAG AAGGAAGGAA GAGAAAGAAT TATAAGGAAG AG - #AAAATATA       8760                                                                          - TTTACTATTG ATAAAGTGGA AGTGGATCAT CATAAAGGTG TTCATCCTCG TC - #ATCTTCAT       8820                                                                          - GTTGAGTAGG CTGAGGAGGA GGAGGAGGAG GAAGAGCAGG GGCCACGGCA GG - #AGAAAAGA       8880                                                                          - TGGAGGAAGT AGGAGGCGGC ACACTTGGTG TAACTTTTAT TTAAAAAAAT TT - #GCATACAA       8940                                                                          - GTGGATCCAC AGAGTTCAAA CCCATGTTGT TCAGGGGTCA ACTGTCTTTG GT - #TAAATAAA       9000                                                                          - ATATATTATT AAAATTAATT TCACCTGTTC CTTTTTACTT TTTCTAATGT GA - #CTACTAGA       9060                                                                          - AAACTTAAAA TGACATCTGA GGCTCCATTG TCTTCCCCTT GGGCCAGCAC TA - #CCACAGAA       9120                                                                          - TGTCTTAGGA TTCAGCTCCA GGCCGCCACG CCTGCTTCTT TCAGGGAGCT GG - #TTCTATGC       9180                                                                          - ACATGTTTTA TATGAGAGAT AATTAAGTTG TCAATTGTGA TAACAAAACA GG - #ATTTGACT       9240                                                                          - TTGTACAGAA TTCTTTGGTT CCAACCAAGC TCATTTCCTT TGTTTCAGCA AA - #CCTCGGAC       9300                                                                          - AGCCAACAAT TCAGAGTTTT GAACAGGTGG GAACAAAAGT GAATGTGACC GT - #AGAAGATG       9360                                                                          - AACGGACTTT AGTCAGAAGG AACAACACTT TCCTAAGCCT CCGGGATGTT TT - #TGGCAAGG       9420                                                                          - ACTTAATTTA TACACTTTAT TATTGGAAAT CTTCAAGTTC AGGAAAGGTG AG - #CATTTTTT       9480                                                                          - AATTTGTTTT TATGACCTGT TTTAAATTGT GAATACTTGG TTTTACAACC CA - #TTTCTTCC       9540                                                                          - CCAATTCAAA AATAGCAGAA CAGAGTTGTT GAGAAGGTGA TGGAGTAGAA GG - #GGGAGCGC       9600                                                                          - GCACTGTGGG GAGGGGTGGA CAACAGGCCT GGTCCTACCT GTGACTCTGC AC - #TACCCTGT       9660                                                                          - GACTCTGGCA GGGCCCCCTC GGAGACCCAG GTTCCTCAGC CAACCGGCTG GA - #TCAGGTCA       9720                                                                          - TCTCTAAAGG TCCCGCCACG CTCACATTTC TCCCTCTATT GAGGATCCCA GG - #CACAAAAT       9780                                                                          - TTGTTTTTGG TTCAATGCAT AATACTCCCT TCCTTTTTCT TTTACTGCAG AT - #ATCTTCTA       9840                                                                          - AAGGGGCTCA ATAGGGTTCA ATATGCCTAA ATTGGATCTT CTCAGTCTTG GA - #AAAGGCAT       9900                                                                          - TTTTAGCAGT GATCAAGGGA AACTGATTAG CGAAGTCACT TCTAATCCTT CA - #CGTGTCAG       9960                                                                          - CTGTGTTCTT GTAGGCTTTG CTTAGAACCT AGGTTTTTAC TTCCACAGTG AC - #TTAATAAA       10020                                                                         - GGGGAAAGAA TTGACTCAGA GCCCAGATGA ATTAAGAACT CTATCTTTTT AC - #AGAAAACA       10080                                                                         - GCCAAAACAA ACACTAATGA GTTTTTGATT GATGTGGATA AAGGAGAAAA CT - #ACTGTTTC       10140                                                                         - AGTGTTCAAG CAGTGATTCC CTCCCGAACA GTTAACCGGA AGAGTACAGA CA - #GCCCGGTA       10200                                                                         - GAGTGTATGG GCCAGGAGAA AGGGGAATTC AGAGGTGAGT GGCTCTGCCA GC - #CATTTGCC       10260                                                                         - TGGGGGTATG GGTGCTGTGG GTGACTTCTG GAGGAGTAGC TCCACCCTCA GG - #GCTGGGAT       10320                                                                         - ATACTTCCTT GGTTAAATAT TCAGGAAAAC AAACTGCCTG GAGGTTTTTT GT - #TGTTATTT       10380                                                                         - GTTTGTTTTG GTTTTGATTT TGCTTTGGTA CAAAAAAGAT TTTGGACATT TA - #GAAATGTT       10440                                                                         - TCTGTGTTGA TTGTGCCCTT GTATTAGCAG GTGTTTTCTT GAGCACCTGT CA - #TGTGCTAA       10500                                                                         - GCCCTCTGCT GAGCACTGGA TACACAAACT GTGTTTAGGA TTTAGCAACA AG - #TCACAGAT       10560                                                                         - TTCCCTGGGC ATTTTTTCAT GCTTAAATTC TAATTCTGGG GGTGGCTTCT GG - #ACCAGCTG       10620                                                                         - CAACAGGACA CAGTAGACAT TCGTGAGTAC CCACTGTGGG CTGTTGCCAC AG - #AGGCTGTA       10680                                                                         - GAGTCTAACC CATCAAGGGA AGGGATTGAG TATATCAAAT ATACCCACAT GC - #ATGCATGT       10740                                                                         - GTGTATATGG CGGACACGTG TGTGTACATG CATGTGCATA TGTTGGGAGC TC - #AGGCCCAT       10800                                                                         - TGTGCGAGGA ACAGTCCCTA ACCGGAAGTG CTGTGGGCCT TCAGACTCTT GC - #AGGAAGCT       10860                                                                         - GCAAGCCTGT GTGTCTCGAT CCATGCCTTA CAGGGAAAGT ATTCTGAGTA CT - #TTCAGTGA       10920                                                                         - AGAAAAGAGT CAGGGGATAT AAACGATGGC TTACGCTGGG TGTGGTGGCT CA - #CGCCTGTA       10980                                                                         - GTCCCTGCAC TTTGGGAGGC CCAGACAGGC AAATCACTTG AGGTCAGGAG TT - #TGGGACCA       11040                                                                         - GCCTGGCCAA CATGGTAAAA GCCCATCTCT ACTCAAAATA CAAAAAGTAG CT - #GGGTGTGG       11100                                                                         - TTGCACGTGT CTGTAGTCCC AGCTACTCAG GAGGTTGAGG CAGGAGAATT GC - #TTGAACCT       11160                                                                         - GGGAGGCGGA GGCTGAAGTG AGCTGAGATT GGACCACTGT ACTCCAGCCT GG - #GTGACAGA       11220                                                                         - GCGAGATTCC ATCTCAAAAA AAAAAAAAAG AAACAACGAA AAAAGAAATG AT - #GGCTTAGC       11280                                                                         - TCCATGTGAA GATGATATTT GAACATTTTA AAACACTTTA AATAAACTGT TC - #TCTCCTGT       11340                                                                         - TTATTGCCAC TGACAGGAGA GGTTTCTCTT TACCTCTGGT CCTGCACCCC TC - #TGAGCCAT       11400                                                                         - CCTACCCACA GCCTTCAGTC ATTGTCCTAA AGCCTAGCTC TAATTCCACT GC - #CTCTCCTT       11460                                                                         - TTGTGCACAC ACACTTCTCT GCTTCCCTGG CCGTTCTCTA TCTTGGAGAG GC - #ATTTCAAA       11520                                                                         - CGCCACTTCC ACCAGAAGGC CTTGCTACTG CACCAACTAG TTACTATCTC TT - #CTTCACCC       11580                                                                         - AAATCCTGGT AGCACTTTGG ATCTCCCACT TGCACTTAGG GTTCACCTTC CG - #TTATAATC       11640                                                                         - ATTGCCATCA ATCTCAGCAT CGTTTTAGGC ACTTCTTTCC AGCCATTGTT CT - #TACCTCCA       11700                                                                         - ACTACATATC TTTTCTGGAC TGTGCATTAT TCAGTTTATT AAATGCCCAT TA - #AATGTGTT       11760                                                                         - TAGCCATTGT CAATTACTCT GAAACGTTCA GGTTTTGACA AATTCTTTCC TA - #ATGTAAGT       11820                                                                         - GTGGTGGAAA GAGTGAAAGA AAGTCAAATT GCACAAAAAT AGGATGGTGT AA - #TTTGGGGT       11880                                                                         - TATGCCGTCA ATTTTGTCCA CTGATAAATG GGATTTGAGC TCTCCAAGTT GA - #CTAGATGC       11940                                                                         - CCTTTATTTT TCAGAAATAT TCTACATCAT TGGAGCTGTG GTATTTGTGG TC - #ATCATCCT       12000                                                                         - TGTCATCATC CTGGCTATAT CTCTACACAA GTGTAGAAAG GCAGGAGTGG GG - #CAGAGCTG       12060                                                                         - GAAGGAGAAC TCCCCACTGA ATGTTTCATA AAGGAAGCAC TGTTGGAGCT AC - #TGCAAATG       12120                                                                         - CTATATTGCA CTGTGACCGA GAACTTTTAA GAGGATAGAA TACATGGAAA CG - #CAAATGAG       12180                                                                         - TATTTCGGAG CATGAAGACC CTGGAGTTCA AAAAACTCTT GATATGACCT GT - #TATTACCA       12240                                                                         - TTAGCATTCT GGTTTTGACA TCAGCATTAG TCACTTTGAA ATGTAACGAA TG - #GTACTACA       12300                                                                         - ACCAATTCCA AGTTTTAATT TTTAACACCA TGGCACCTTT TGCACATAAC AT - #GCTTTAGA       12360                                                                         - TTATATATTC CGCACTCAAG GAGTAACCAG GTCGTCCAAG CAAAAACAAA TG - #GGAAAATG       12420                                                                         - TCTTAAAAAA TCCTGGGTGG ACTTTTGAAA AGCTTTTTTT TTTTTTTTTT TT - #TTTGAGAC       12480                                                                         - GGAGTCTTGC TCTGTTGCCC AGGCTGGAGT GCAGTAGCAC GATCTCGGCT CA - #CTGCACCC       12540                                                                         - TCCGTCTCTC GGGTTCAAGC AATTGTCTGC CTCAGCCTCC CGAGTAGCTG GG - #ATTACAGG       12600                                                                         - TGCGCACTAC CACGCCAAGC TAATTTTTGT ATTTTTTAGT AGAGATGGGG TT - #TCACCATC       12660                                                                         - TTGGCCAGGC TGGTCTTGAA TTCCTGACCT CAGGTGATCC ACCCACCTTG GC - #CTCCCAAA       12720                                                                         - GTGCTAGTAT TATGGGCGTG AACCACCATG CCCAGCCGAA AAGCTTTTGA GG - #GGCTGACT       12780                                                                         - TCAATCCATG TAGGAAAGTA AAATGGAAGG AAATTGGGTG CATTTCTAGG AC - #TTTTCTAA       12840                                                                         - CATATGTCTA TAATATAGTG TTTAGGTTCT TTTTTTTTTC AGGAATACAT TT - #GGAAATTC       12900                                                                         - AAAACAATTG GCAAACTTTG TATTAATGTG TTAAGTGCAG GAGACATTGG TA - #TTCTGGGC       12960                                                                         - ACCTTCCTAA TATGCTTTAC AATCTGCACT TTAACTGACT TAAGTGGCAT TA - #AACATTTG       13020                                                                         - AGAGCTAACT ATATTTTTAT AAGACTACTA TACAAACTAC AGAGTTTATG AT - #TTAAGGTA       13080                                                                         - CTTAAAGCTT CTATGGTTGA CATTGTATAT ATAATTTTTT AAAAAGGTTT TC - #TATATGGG       13140                                                                         - GATTTTCTAT TTATGTAGGT AATATTGTTC TATTTGTATA TATTGAGATA AT - #TTATTTAA       13200                                                                         - TATACTTTAA ATAAAGGTGA CTGGGAATTG TTACTGTTGT ACTTATTCTA TC - #TTCCATTT       13260                                                                         - ATTATTTATG TACAATTTGG TGTTTGTATT AGCTCTACTA CAGTAAATGA CT - #GTAAAATT       13320                                                                         - GTCAGTGGCT TACAACAACG TATCTTTTTC GCTTATAATA CATTTTGGTG AC - #TGTAGGCT       13380                                                                         - GACTGCACTT CTTCTCAATG TTTTCTCATT CTAGGATGCA AACCAATGGA GA - #AGCCCCTA       13440                                                                         - ATTAGATCAG GGCAGAGGGA AAAACAAAAA ACTGGTAGAA ACCGGCAACC AC - #AGCTTCAA       13500                                                                         - GCTTTAAGCC CATCTCCTAC ACTTCTGCTC TGTACGTGCC CATTGTCACT TC - #TGTTCACA       13560                                                                         - TGCTACTGTC CCAAGCAAGT GACCAAGCCT GACAATACTT TGTCTACTGG AG - #TCACTGCA       13620                                                                         - AGGCACATGA CGGGGCAGGG ATGTCGTCTT ACAGGGAAGA GAAAAGATAA TG - #CTCTCTAC       13680                                                                         - TGCAGACTTG GAGAGATTTC TTCCCATTGG CAGTAGTTTG ACTAATTGGA GA - #TGAGAAAA       13740                                                                         - AAAGAAACAT TCTTGGGATG ATTGTATTGA AACAAAATTA GGTAAAAGGA CA - #ATATAGGA       13800                                                                         - TAGGGAGAGA TATAAGTGGA ATGAGATCTC TAGAGTCCAT TAAAAGCAAG CT - #AGATTGAG       13860                                                                         #         13865                                                               - (2) INFORMATION FOR SEQ ID NO:12:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 263 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                - Ser Gly Thr Thr Asn Thr Val Ala Ala Tyr As - #n Leu Thr Trp Lys Ser         #                15                                                           - Thr Asn Phe Lys Thr Ile Leu Glu Trp Glu Pr - #o Lys Pro Val Asn Gln         #            30                                                               - Val Tyr Thr Val Gln Ile Ser Thr Lys Ser Gl - #y Asp Trp Lys Ser Lys         #        45                                                                   - Cys Phe Tyr Thr Thr Asp Thr Glu Cys Asp Le - #u Thr Asp Glu Ile Val         #    60                                                                       - Lys Asp Val Lys Gln Thr Tyr Leu Ala Arg Va - #l Phe Ser Tyr Pro Ala         #80                                                                           - Gly Asn Val Glu Ser Thr Gly Ser Ala Gly Gl - #u Pro Leu Tyr Glu Asn         #                95                                                           - Ser Pro Glu Phe Thr Pro Tyr Leu Glu Thr As - #n Leu Gly Gln Pro Thr         #           110                                                               - Ile Gln Ser Phe Glu Gln Val Gly Thr Lys Va - #l Asn Val Thr Val Glu         #       125                                                                   - Asp Glu Arg Thr Leu Val Arg Arg Asn Asn Th - #r Phe Leu Ser Leu Arg         #   140                                                                       - Asp Val Phe Gly Lys Asp Leu Ile Tyr Thr Le - #u Tyr Tyr Trp Lys Ser         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Ser Ser Ser Gly Lys Lys Thr Ala Lys Thr As - #n Thr Asn Glu Phe Leu         #               175                                                           - Ile Asp Val Asp Lys Gly Glu Asn Tyr Cys Ph - #e Ser Val Gln Ala Val         #           190                                                               - Ile Pro Ser Arg Thr Val Asn Arg Lys Ser Th - #r Asp Ser Pro Val Glu         #       205                                                                   - Cys Met Gly Gln Glu Lys Gly Glu Phe Arg Gl - #u Ile Phe Tyr Ile Ile         #   220                                                                       - Gly Ala Val Val Phe Val Val Ile Ile Leu Va - #l Ile Ile Leu Ala Ile         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Ser Leu His Lys Cys Arg Lys Ala Gly Val Gl - #y Gln Ser Trp Lys Glu         #               255                                                           - Asn Ser Pro Leu Asn Val Ser                                                             260                                                               - (2) INFORMATION FOR SEQ ID NO:13:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 1440 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                - TCAACAGGCA GGGGCAGCAC TGCAGAGATT TCATCATGGT CTCCCAGGCC CT - #CAGGCTCC         60                                                                          - TCTGCCTTCT GCTTGGGCTT CAGGGCTGCC TGGCTGCAGG CGGGGTCGCT AA - #GGCCTCAG        120                                                                          - GAGGAGAAAC ACGGGACATG CCGTGGAAGC CGGGGCCTCA CAGAGTCTTC GT - #AACCCAGG        180                                                                          - AGGAAGCCCA CGGCGTCCTG CACCGGCGCC GGCGCGCCAA CGCGTTCCTG GA - #GGAGCTGC        240                                                                          - GGCCGGGCTC CCTGGAGAGG GAGTGCAAGG AGGAGCAGTG CTCCTTCGAG GA - #GGCCCGGG        300                                                                          - AGATCTTCAA GGACGCGGAG AGGACGAAGC TGTTCTGGAT TTCTTACAGT GA - #TGGGGACC        360                                                                          - AGTGTGCCTC AAGTCCATGC CAGAATGGGG GCTCCTGCAA GGACCAGCTC CA - #GTCCTATA        420                                                                          - TCTGCTTCTG CCTCCCTGCC TTCGAGGGCC GGAACTGTGA GACGCACAAG GA - #TGACCAGC        480                                                                          - TGATCTGTGT GAACGAGAAC GGCGGCTGTG AGCAGTACTG CAGTGACCAC AC - #GGGCACCA        540                                                                          - AGCGCTCCTG TCGGTGCCAC GAGGGGTACT CTCTGCTGGC AGACGGGGTG TC - #CTGCACAC        600                                                                          - CCACAGTTGA ATATCCATGT GGAAAAATAC CTATTCTAGA AAAAAGAAAT GC - #CAGCAAAC        660                                                                          - CCCAAGGCCG AATTGTGGGG GGCAAGGTGT GCCCCAAAGG GGAGTGTCCA TG - #GCAGGTCC        720                                                                          - TGTTGTTGGT GAATGGAGCT CAGTTGTGTG GGGGGACCCT GATCAACACC AT - #CTGGGTGG        780                                                                          - TCTCCGCGGC CCACTGTTTC GACAAAATCA AGAACTGGAG GAACCTGATC GC - #GGTGCTGG        840                                                                          - GCGAGCACGA CCTCAGCGAG CACGACGGGG ATGAGCAGAG CCGGCGGGTG GC - #GCAGGTCA        900                                                                          - TCATCCCCAG CACGTACGTC CCGGGCACCA CCAACCACGA CATCGCGCTG CT - #CCGCCTGC        960                                                                          - ACCAGCCCGT GGTCCTCACT GACCATGTGG TGCCCCTCTG CCTGCCCGAA CG - #GACGTTCT       1020                                                                          - CTGAGAGGAC GCTGGCCTTC GTGCGCTTCT CATTGGTCAG CGGCTGGGGC CA - #GCTGCTGG       1080                                                                          - ACCGTGGCGC CACGGCCCTG GAGCTCATGG TGCTCAACGT GCCCCGGCTG AT - #GACCCAGG       1140                                                                          - ACTGCCTGCA GCAGTCACGG AAGGTGGGAG ACTCCCCAAA TATCACGGAG TA - #CATGTTCT       1200                                                                          - GTGCCGGCTA CTCGGATGGC AGCAAGGACT CCTGCAAGGG GGACAGTGGA GG - #CCCACATG       1260                                                                          - CCACCCACTA CCGGGGCACG TGGTACCTGA CGGGCATCGT CAGCTGGGGC CA - #GGGCTGCG       1320                                                                          - CAACCGTGGG CCACTTTGGG GTGTACACCA GGGTCTCCCA GTACATCGAG TG - #GCTGCAAA       1380                                                                          - AGCTCATGCG CTCAGAGCCA CGCCCAGGAG TCCTCCTGCG AGCCCCATTT CC - #CTAGCCCA       1440                                                                          - (2) INFORMATION FOR SEQ ID NO:14:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 466 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                - Met Val Ser Gln Ala Leu Arg Leu Leu Cys Le - #u Leu Leu Gly Leu Gln         #                15                                                           - Gly Cys Leu Ala Ala Gly Gly Val Ala Lys Al - #a Ser Gly Gly Glu Thr         #            30                                                               - Arg Asp Met Pro Trp Lys Pro Gly Pro His Ar - #g Val Phe Val Thr Gln         #        45                                                                   - Glu Glu Ala His Gly Val Leu His Arg Arg Ar - #g Arg Ala Asn Ala Phe         #    60                                                                       - Leu Glu Glu Leu Arg Pro Gly Ser Leu Glu Ar - #g Glu Cys Lys Glu Glu         #80                                                                           - Gln Cys Ser Phe Glu Glu Ala Arg Glu Ile Ph - #e Lys Asp Ala Glu Arg         #                95                                                           - Thr Lys Leu Phe Trp Ile Ser Tyr Ser Asp Gl - #y Asp Gln Cys Ala Ser         #           110                                                               - Ser Pro Cys Gln Asn Gly Gly Ser Cys Lys As - #p Gln Leu Gln Ser Tyr         #       125                                                                   - Ile Cys Phe Cys Leu Pro Ala Phe Glu Gly Ar - #g Asn Cys Glu Thr His         #   140                                                                       - Lys Asp Asp Gln Leu Ile Cys Val Asn Glu As - #n Gly Gly Cys Glu Gln         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Tyr Cys Ser Asp His Thr Gly Thr Lys Arg Se - #r Cys Arg Cys His Glu         #               175                                                           - Gly Tyr Ser Leu Leu Ala Asp Gly Val Ser Cy - #s Thr Pro Thr Val Glu         #           190                                                               - Tyr Pro Cys Gly Lys Ile Pro Ile Leu Glu Ly - #s Arg Asn Ala Ser Lys         #       205                                                                   - Pro Gln Gly Arg Ile Val Gly Gly Lys Val Cy - #s Pro Lys Gly Glu Cys         #   220                                                                       - Pro Trp Gln Val Leu Leu Leu Val Asn Gly Al - #a Gln Leu Cys Gly Gly         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Thr Leu Ile Asn Thr Ile Trp Val Val Ser Al - #a Ala His Cys Phe Asp         #               255                                                           - Lys Ile Lys Asn Trp Arg Asn Leu Ile Ala Va - #l Leu Gly Glu His Asp         #           270                                                               - Leu Ser Glu His Asp Gly Asp Glu Gln Ser Ar - #g Arg Val Ala Gln Val         #       285                                                                   - Ile Ile Pro Ser Thr Tyr Val Pro Gly Thr Th - #r Asn His Asp Ile Ala         #   300                                                                       - Leu Leu Arg Leu His Gln Pro Val Val Leu Th - #r Asp His Val Val Pro         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Leu Cys Leu Pro Glu Arg Thr Phe Ser Glu Ar - #g Thr Leu Ala Phe Val         #               335                                                           - Arg Phe Ser Leu Val Ser Gly Trp Gly Gln Le - #u Leu Asp Arg Gly Ala         #           350                                                               - Thr Ala Leu Glu Leu Met Val Leu Asn Val Pr - #o Arg Leu Met Thr Gln         #       365                                                                   - Asp Cys Leu Gln Gln Ser Arg Lys Val Gly As - #p Ser Pro Asn Ile Thr         #   380                                                                       - Glu Tyr Met Phe Cys Ala Gly Tyr Ser Asp Gl - #y Ser Lys Asp Ser Cys         385                 3 - #90                 3 - #95                 4 -       #00                                                                           - Lys Gly Asp Ser Gly Gly Pro His Ala Thr Hi - #s Tyr Arg Gly Thr Trp         #               415                                                           - Tyr Leu Thr Gly Ile Val Ser Trp Gly Gln Gl - #y Cys Ala Thr Val Gly         #           430                                                               - His Phe Gly Val Tyr Thr Arg Val Ser Gln Ty - #r Ile Glu Trp Leu Gln         #       445                                                                   - Lys Leu Met Arg Ser Glu Pro Arg Pro Gly Va - #l Leu Leu Arg Ala Pro         #   460                                                                       - Phe Pro                                                                     465                                                                           - (2) INFORMATION FOR SEQ ID NO:15:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 27 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                #             27   AGGC ACTACAA                                               - (2) INFORMATION FOR SEQ ID NO:16:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 31 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                #          31      TGAA TTCCCCTTTC T                                          - (2) INFORMATION FOR SEQ ID NO:17:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 47 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                #                47GGTG CCTCGTGCTT CTGGCACTAC AAATACT                         - (2) INFORMATION FOR SEQ ID NO:18:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 50 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                #              50CTGGTG CCTCGTGGTT CTTGCGGCAC TACAAATACT                      - (2) INFORMATION FOR SEQ ID NO:19:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 53 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                - CGCGGATCCA CCGCCACCAG ATCCACCGCC TCCTTCTCTG AATTCCCCTT TC - #T                53                                                                          - (2) INFORMATION FOR SEQ ID NO:20:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 44 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                # 44               GAGG CTCTTCAGGC ACTACAAATA CTGT                            - (2) INFORMATION FOR SEQ ID NO:21:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 31 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                #          31      TGAA TTCCCCTTTC T                                          - (2) INFORMATION FOR SEQ ID NO:22:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 50 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                #              50CTGGTG CCTCGTGGTT CTTGCGGCAC TACAAATACT                      - (2) INFORMATION FOR SEQ ID NO:23:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 31 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                #          31      TGAA TTCCCCTTTC T                                          - (2) INFORMATION FOR SEQ ID NO:24:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 44 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                # 44               GGTG CCTCGTGGTT GCACTACAAA TACT                            - (2) INFORMATION FOR SEQ ID NO:25:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 34 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                #        34        CTCT GAATTCCCCT TTCT                                       - (2) INFORMATION FOR SEQ ID NO:26:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 19 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                # 19               AAC                                                        - (2) INFORMATION FOR SEQ ID NO:27:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 36 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                #       36         AATC TTCAGCTTCA GGAAAG                                     __________________________________________________________________________

What is claimed is:
 1. A method for treating an animal having avascularized tumor, comprising administering to said animal at least afirst coagulation-deficient Tissue Factor compound and at least a firstanti-cancer agent in a combined amount effective to promote coagulationin the tumor vasculature and to induce tumor necrosis.
 2. The method ofclaim 1, wherein said coagulation-deficient Tissue Factor compound isadministered to said animal systemically.
 3. The method of claim 2,wherein said coagulation-deficient Tissue Factor compound isadministered to said animal via intravenous injection.
 4. The method ofclaim 1, wherein said anti-cancer agent is administered to said animalvia intravenous injection.
 5. The method of claim 1, wherein saidcoagulation-deficient Tissue Factor compound and said anti-cancer agentare administered to said animal simultaneously.
 6. The method of claim5, wherein a single composition comprising said coagulation-deficientTissue Factor compound and said anti-cancer agent is administered tosaid animal.
 7. The method of claim 1, wherein saidcoagulation-deficient Tissue Factor compound is administered to saidanimal at a biologically effective time prior to administration of saidanti-cancer agent.
 8. The method of claim 1, wherein said anti-canceragent is administered to said animal at a biologically effective timeprior to administration of said coagulation-deficient Tissue Factorcompound.
 9. The method of claim 1, wherein said anti-cancer agent is achemotherapeutic agent.
 10. The method of claim 9, wherein saidanti-cancer agent is a chemotherapeutic agent listed in Table II. 11.The method of claim 10, wherein said anti-cancer agent is etoposide. 12.The method of claim 1, wherein said anti-cancer agent is an antibodyconstruct comprising an antibody or antigen binding region thatspecifically binds to a component of a tumor cell, tumor vasculature ortumor stroma, the antibody operatively attached to a cytotoxic agent orto a coagulation factor.
 13. The method of claim 12, wherein saidantibody construct comprises an antibody or antigen binding region thatspecifically binds to a component of tumor vasculature.
 14. The methodof claim 12, wherein said antibody construct comprises an antibody orantigen binding region that specifically binds to a component of tumorstroma.
 15. The method of claim 12, wherein said antibody constructcomprises an antibody or antigen binding region operatively attached toa cytotoxic agent.
 16. The method of claim 12, wherein said antibodyconstruct comprises an antibody or antigen binding region operativelyattached to a coagulant.
 17. The method of claim 16, wherein saidantibody construct comprises an antibody or antigen binding regionoperatively attached to Tissue Factor or a Tissue Factor derivative. 18.The method of claim 1, wherein said coagulation-deficient Tissue Factorcompound is between about 100-fold and about 1,000,000-fold less activein coagulation than full length, native Tissue Factor.
 19. The method ofclaim 18, wherein said coagulation-deficient Tissue Factor compound isat least about 1,000-fold less active in coagulation than full length,native Tissue Factor.
 20. The method of claim 19, wherein saidcoagulation-deficient Tissue Factor compound is at least about10,000-fold less active in coagulation than full length, native TissueFactor.
 21. The method of claim 20, wherein said coagulation-deficientTissue Factor compound is at least about 100,000-fold less active incoagulation than full length, native Tissue Factor.
 22. The method ofclaim 1, wherein said coagulation-deficient Tissue Factor compound is ahuman Tissue Factor compound.
 23. The method of claim 1, wherein saidcoagulation-deficient Tissue Factor compound is prepared by recombinantexpression.
 24. The method of claim 1, wherein saidcoagulation-deficient Tissue Factor compound is a truncated TissueFactor.
 25. The method of claim 24, wherein said coagulation-deficientTissue Factor compound consists essentially of the amino acid sequenceof SEQ ID NO:1.
 26. The method of claim 1, wherein saidcoagulation-deficient Tissue Factor compound is a dimeric Tissue Factor.27. The method of claim 1, wherein said coagulation-deficient TissueFactor compound is a mutant Tissue Factor deficient in the ability toactivate Factor VII.
 28. The method of claim 27, further comprisingadministering to said animal an amount of Factor VIIa sufficient toincrease tumor vasculature coagulation and tumor necrosis.
 29. Themethod of claim 1, further comprising administering to said animal atherapeutically effective amount of at least one of Factor VIIa or anactivator of Factor VII.
 30. The method of claim 1, wherein saidcoagulation-deficient Tissue Factor compound has been modified toincrease its biological half life.
 31. The method of claim 1, wherein atleast a second coagulation-deficient Tissue Factor compound isadministered to said animal, said second coagulation-deficient TissueFactor compound being of a distinct type from said firstcoagulation-deficient Tissue Factor compound.
 32. The method of claim31, wherein said first and second coagulation-deficient Tissue Factorcompounds are distinct types of coagulation-deficient Tissue Factorcompounds selected from the group consisting of truncated, mutant,homodimeric, heterodimeric, polymeric, multimeric and increasedbiological half life coagulation-deficient Tissue Factor compounds. 33.The method of claim 1, wherein said animal has a vascularized tumor ofat least about medium size.
 34. The method of claim 33, wherein saidanimal has a large vascularized tumor.
 35. The method of claim 1,wherein said animal is a human subject.
 36. The method of claim 1,wherein at least a second anti-cancer agent is administered to saidanimal.
 37. The method of claim 36, wherein said second anti-canceragent is of a type distinct from said first anti-cancer agent.
 38. Themethod of claim 1, wherein said anti-cancer agent is a radiotherapeuticagent.
 39. The method of claim 1, wherein said anti-cancer agent is anagent that induces apoptosis.
 40. The method of claim 1, wherein saidanti-cancer agent is a cytokine.
 41. A method for treating an animalhaving a vascularized tumor, comprising administering to said animal atleast a first coagulation-impaired Tissue Factor compound and at least afirst anti-cancer agent in a combined amount effective to promotecoagulation in the tumor vasculature and to induce tumor necrosis.
 42. Amethod for treating an animal having a vascularized tumor, comprisingadministering to said animal at least a first coagulation-impairedTissue Factor compound and at least a first anti-cancer agent in acombined amount effective to achieve coagulation and tissue necrosisthat are substantially confined to the vascularized tumor and do notsubstantially extend to normal, healthy tissues.
 43. A method fortreating an animal having a vascularized tumor, comprising administeringto said animal at least a first Tissue Factor compound and at least afirst anti-cancer agent in a combined amount effective to promotecoagulation in the tumor vasculature and to induce tumor necrosis;wherein said at least a first Tissue Factor compound is renderedcoagulation-deficient by being deficient in binding to a phospholipidsurface or deficient in inserting into a phospholipid membrane or lipidbilayer.
 44. A method for treating an animal having a vascularizedtumor, comprising administering to said animal at least a first TissueFactor compound and at least a first anti-cancer agent in a combinedamount effective to promote coagulation in the tumor vasculature and toinduce tumor necrosis; wherein said at least a first Tissue Factorcompound is rendered coagulation-deficient by including a mutation thatconfers a deficiency in the ability to activate Factor VII.
 45. A methodfor treating an animal having a vascularized tumor, comprisingadministering to said animal at least a first coagulation-deficientTissue Factor compound and at least a first anti-cancer agent in acombined amount effective to promote coagulation in the tumorvasculature and to induce tumor necrosis; wherein saidcoagulation-deficient Tissue Factor compound substantially retains itsability to bind to Factor VII or Factor VIIa and is renderedcoagulation-deficient by being deficient in binding to a phospholipidsurface, deficient in inserting into a phospholipid membrane ordeficient in activating Factor VII.
 46. A method for treating an animalhaving a vascularized tumor, comprising administering to said animal atleast a first coagulation-deficient Tissue Factor compound and at leasta first anti-cancer agent in a combined amount effective to promotecoagulation in the tumor vasculature and to induce tumor necrosis;wherein said coagulation-deficient Tissue Factor compound is renderedcoagulation-deficient by being deficient in binding to a phospholipidsurface, deficient in inserting into a phospholipid membrane ordeficient in activating Factor VII and yet retains the catalyticactivity of activating Factor X in the presence of Factor VIIa.